Diagnosis and treatment of malignant neoplasms

ABSTRACT

The invention features a method for diagnosing a malignant neoplasm in a mammal by contacting a bodily fluid from the mammal with an antibody which binds to an human aspartyl (asparaginyl) beta-hydroxylase (HAAH) polypeptide and methods of treating malignant neoplasms by inhibiting HAAH.

DIAGNOSIS AND TREATMENT OF MALIGNANT NEOPLASMS Statement as to FederallySponsored Research

[0001] This invention was made with U.S. Government support underNational Institutes of Health grants CA-35711, AA-02666, AA-02169, andAA11431. The government has certain rights in the invention.

BACKGROUND OF THE INVENTION

[0002] Primary malignant central nervous system (CNS) neoplasms,particularly glioblastomas, are highly fatal due to their aggressive andwidespread infiltration of the brain and resistance to anti-cancertreatments. Although progress has been made in unraveling thepathological mechanisms underlying CNS cancers as well as other cancertypes, tumor specific therapeutic approaches and methods of diagnosishave been largely elusive.

SUMMARY OF THE INVENTION

[0003] The invention features a method for diagnosing a malignantneoplasm in a mammal by contacting a bodily fluid from the mammal withan antibody which binds to an human aspartyl (asparaginyl)beta-hydroxylase (HAAH) polypeptide under conditions sufficient to forman antigen-antibody complex and detecting the antigen-antibody complex.Malignant neoplasms detected in this manner include those derived fromendodermal tissue, e.g., colon cancer, breast cancer, pancreatic cancer,liver cancer, and cancer of the bile ducts. Neoplasms of the centralnervous system (CNS) such as primary malignant CNS neoplasms of bothneuronal and glial cell origin and metastatic CNS neoplasms are alsodetected. Patient derived tissue samples, e.g., biopsies of solidtumors, as well as bodily fluids such as a CNS-derived bodily fluid,blood, serum, urine, saliva, sputum, lung effusion, and ascites fluid,are contacted with an HAAH-specific antibody.

[0004] The assay format is also useful to generate temporal data usedfor prognosis of malignant disease. A method for prognosis of amalignant neoplasm of a mammal is carried out by (a) contacting a bodilyfluid from the mammal with an antibody which binds to an HAAHpolypeptide under conditions sufficient to form an antigen-antibodycomplex and detecting the antigen-antibody complex; (b) quantitating theamount of complex to determine the level of HAAH in the fluid; and (c)comparing the level of HAAH in the fluid with a normal control level ofHAAH. An increasing level of HAAH over time indicates a progressiveworsening of the disease, and therefore, an adverse prognosis.

[0005] The invention also includes an antibody which binds to HAAH. Theantibody preferably binds to a site in the carboxyterminal catalyticdomain of HAAH. Alternatively, the antibody binds to an epitope that isexposed on the surface of the cell. The antibody is a polyclonalantisera or monoclonal antibody. The invention encompasses not only anintact monoclonal antibody, but also an immunologically-active antibodyfragment, e. g., a Fab or (Fab)₂ fragment; an engineered single chain FVmolecule; or a chimeric molecule, e.g., an antibody which contains thebinding specificity of one antibody, e.g., of murine origin, and theremaining portions of another antibody, e.g., of human origin.Preferably the antibody is a monoclonal antibody such as FB50, 5C7, 5E9,19B, 48A, 74A, 78A, 86A, HA238A, HA221, HA 239, HA241, HA329, or HA355.Antibodies which bind to the same epitopes as those monoclonalantibodies are also within the invention.

[0006] An HAAH-specific intrabody is a recombinant single chainHAAH-specific antibody that is expressed inside a target cell, e.g.,tumor cell. Such an intrabody binds to endogenous intracellular HAAH andinhibits HAAH enzymatic activity or prevents HAAH from binding to anintracellular ligand. HAAH-specific intrabodies inhibit intracellularsignal transduction, and as a result, inhibit growth of tumors whichoverexpress HAAH.

[0007] A kit for diagnosis of a tumor in a mammal contains anHAAH-specific antibody. The diagnostic assay kit is preferentiallyformulated in a standard two-antibody binding format in which oneHAAH-specific antibody captures HAAH in a patient sample and anotherHAAH-specific antibody is used to detect captured HAAH. For example, thecapture antibody is immobilized on a solid phase, e.g., an assay plate,an assay well, a nitrocellulose membrane, a bead, a dipstick, or acomponent of an elution column. The second antibody, i.e., the detectionantibody, is typically tagged with a detectable label such as acalorimetric agent or radioisotope.

[0008] Also within the invention is a method of inhibiting tumor growthin a mammal, which is carried out by administering to the mammal acompound which inhibits expression or enzymatic activity of HAAH.Preferably, the compound is substantially pure nucleic acid moleculesuch as an HAAH antisense DNA, the sequence of which is complementary toa coding sequence of HAAH. Expression of HAAH is inhibited by contactingmammalian cells, e.g., tumor cells, with HAAH antisense DNA or RNA,e.g., a synthetic HAAH antisense oligonucleotide. For example, HAAHantisense nucleic acid is introduced into glioblastoma cells or othertumor cells which overexpress HAAH. Binding of the antisense nucleicacid to an HAAH transcript in the target cell results in a reduction inHAAH production by the cell. By the term “antisense nucleic acid” ismeant a nucleic acid (RNA or DNA) which is complementary to a portion ofan mRNA, and which hybridizes to and prevents translation of the mRNA.Preferably, the antisense DNA is complementary to the 5′ regulatorysequence or the 5′ portion of the coding sequence of HAAH mRNA (e.g., asequence encoding a signal peptide or a sequence within exon 1 of theHAAH gene). Standard techniques of introducing antisense DNA into thecell may be used, including those in which antisense DNA is a templatefrom which an antisense RNA is transcribed. The method is to treattumors in which expression of HAAH is upregulated, e.g., as a result ofmalignant transformation of the cells. The length of the oligonucleotideis at least 10 nucleotides and may be as long as the naturally-occurringHAAH transcript. Preferably, the length is between 10 and 50nucleotides, inclusive. More preferably, the length is between 10 and 20nucleotides, inclusive.

[0009] By “substantially pure DNA or RNA” is meant that the nucleic acidis free of the genes which, in the naturally-occurring genome of theorganism from which the DNA of the invention is derived, flank a HAAHgene. The term therefore includes, for example, a recombinant nucleicacid which is incorporated into a vector, into an autonomouslyreplicating plasmid or virus, or into the genomic DNA of a procaryote oreucaryote at a site other than its natural site; or which exists as aseparate molecule (e.g., a cDNA or a genomic or cDNA fragment producedby PCR or restriction endonuclease digestion) independent of othersequences. It also includes a recombinant nucleic acid which is part ofa hybrid gene encoding additional polypeptide sequence such as a nucleicacid encoding an chimeric polypeptide, e.g., one encoding an antibodyfragment linked to a cytotoxic polypeptide. Alternatively, HAAHexpression is inhibited by administering a ribozyme or a compound whichinhibits binding of Fos or Jun to an HAAH promoter sequence.

[0010] Compounds, which inhibit an enzymatic activity of HAAH, areuseful to inhibit tumor growth in a mammal. By enzymatic activity ofHAAH is meant hydroxylation of an epidermal growth factor (EGF)-likedomain of a polypeptide. For example an EGF-like domain has theconsensus sequence CX₇CX₄CX₁₀CXCX₈C (SEQ ID NO:1). HAAH hydroxylaseactivity is inhibited intracellularly. For example, a dominant negativemutant of HAAH (or a nucleic acid encoding such a mutant) isadministered. The dominant negative HAAH mutant contains a mutationwhich changes a ferrous iron binding site from histidine of anaturally-occurring HAAH sequence to a non-iron-binding amino acid,thereby abolishing the hydroxylase activity of HAAH. The histidine to bemutated, e.g., deleted or substituted, is located in the carboxyterminalcatalytic domain of HAAH. For example, the mutation is located betweenamino acids 650-700 (such as the His motif, underlined sequence of SEQID NO:2) the native HAAH sequence. For example, the mutation is atresidues 671, 675, 679, or 690 of SEQ ID NO:2. An HAAH-specificintrabody is also useful to bind to HAAH and inhibit intracellular HAAHenzymatic activity, e.g., by binding to an epitope in the catalyticdomain of HAAH. Other compounds such as L-mimosine or hydroxypyridoneare administered directly into a tumor site or systemically to inhibitHAAH hydroxylase activity. Table 1 Amino acid sequence of HAAHMAQRKNAKSS GNSSSSGSGS GSTSAGSSSP GARRETKHGG HKNGRKGGLS GTSFFTWFMV 61(SEQ ID NO:2; IALLGVWTSV AVVWFDLVDY EEVLGKLGIY DADGDGDFDV DDAKVLLGLKERSTSEPAVP 121 PEEAEPHTEP EEQVPVEAEP QNIEDEAKEQ IQSLLHEMVH AEHVEGEDLQQEDGPTGEPQ 181 QEDDEFLMAT DVDDRFETLE PEVSHEETEH SYHVEETVSQ DCNQDMEEMMSEQENPDSSE 241 PVVEDERLHH DTDDVTYQVY EEQAVYEPLE NEGIEITEVT APPEDNPVEDSQVIVEEVSI 301 FPVEEQQEVP PETNRKTDDP EQKAKVKKKK PKLLNKFDKT IKAELDAAEKLRKRGKIEEA 361 VNAFKELVRK YPQSPRARYG KAQCEDDLAE KRRSNEVLRG AIETYQEVASLPDVPADLLK 421 LSLKRRSDRQ QFLGHMRGSL LTLQRLVQLF PNDTSLKNDL GVGYLLIGDNDNAKKVYEEV 481 LSVTPNDGFA KVHYGFILKA QNKIAESIPY LKEGIESGDP GTDDGRFYFHLGDAMQRVGN 541 KEAYKWYELG HKRGHFASVW QRSLYNVNGL KAQPWWTPKE TGYTELVKSLERNWKLIRDE 601 GLAVMDKAKG LFLPEDENLR EKGDWSQFTL WQQGRRNENA CKGAPKTCTLLEKFPETTGC 661 RRGQIKYSIM HPGTHVWPHT GPTNCRLRMH LGLVIPKEGC KIRCANETRTWEEGKVLIFD 721 DSFEHEVWQD ASSFRLIFIV DVWHPELTPQ QRRSLPAI

[0011] DSFEHEVWQD ASSFRLIFIV DVWHPELTPQ QRRSLPAI (SEQ ID NO:2; GENBANKAccession No. S83325; His motif is underlined; conserved sequenceswithin the catalytic domain are designated by bold type)

[0012] For example, a compound which inhibits HAAH hydroxylation is apolypeptide that binds a HAAH ligand but does not transduce anintracellular signal or an polypeptide which contains a mutation in thecatalytic site of HAAH. Such a polypeptide contains an amino acidsequence that is at least 50% identical to a naturally-occurring HAAHamino acid sequence or a fragment thereof and which has the ability toinhibit HAAH hydroxylation of substrates containing an EGF-like repeatsequence. More preferably, the polypeptide contains an amino acidsequence that is at least 75%, more preferably at least 85%, morepreferably at least 95% identical to SEQ ID NO:

[0013] A substantially pure HAAH polypeptide or HAAH-derived polypeptidesuch as a mutated HAAH polypeptide is preferably obtained by expressionof a recombinant nucleic acid encoding the polypeptide or by chemicallysynthesizing the protein. A polypeptide or protein is substantially purewhen it is separated from those contaminants which accompany it in itsnatural state (proteins and other naturally-occurring organicmolecules). Typically, the polypeptide is substantially pure when itconstitutes at least 60%, by weight, of the protein in the preparation.Preferably, the protein in the preparation is at least 75%, morepreferably at least 90%, and most preferably at least 99%, by weight,HAAH. Purity is measured by any appropriate method, e.g., columnchromatography, polyacrylamide gel electrophoresis, or HPLC analysis.Accordingly, substantially pure polypeptides include recombinantpolypeptides derived from a eucaryote but produced in E. coli or anotherprocaryote, or in a eucaryote other than that from which the polypeptidewas originally derived.

[0014] Nucleic acid molecules which encode such HAAH or HAAH-derivedpolypeptides are also within the invention. Table 2: HAAH cDNA sequencecggaccgtgc aatggcccag cgtaagaatg ccaagagcag cggcaacagc agcagcagcg 61(SEQ ID NO:3; gctccggcag cggtagcacg agtgcgggca gcagcagccc cggggcccggagagagacaa 121 agcatggagg acacaagaat gggaggaaag gcggactctc gggaacttcattcttcacgt 181 ggtttatggt gattgcattg ctgggcgtct ggacatctgt agctgtcgtttggtttgatc 241 ttgttgacta tgaggaagtt ctaggaaaac taggaatcta tgatgctgatggtgatggag 301 attttgatgt ggatgatgcc aaagttttat taggacttaa agagagatctacttcagagc 361 cagcagtccc gccagaagag gctgagccac acactgagcc cgaggagcaggttcctgtgg 421 aggcagaacc ccagaatatc gaagatgaag caaaagaaca aattcagtcccttctccatg 481 aaatggtaca cgcagaacat gttgagggag aagacttgca acaagaagatggacccacag 541 gagaaccaca acaagaggat gatgagtttc ttatggcgac tgatgtagatgatagatttg 601 agaccctgga acctgaagta tctcatgaag aaaccgagca tagttaccacgtggaagaga 661 cagtttcaca agactgtaat caggatatgg aagagatgat gtctgagcaggaaaatccag 721 attccagtga accagtagta gaagatgaaa gattgcacca tgatacagatgatgtaacat 781 accaagtcta tgaggaacaa gcagtatatg aacctctaga aaatgaagggatagaaatca 841 cagaagtaac tgctccccct gaggataatc ctgtagaaga ttcacaggtaattgtagaag 901 aagtaagcat ttttcctgtg gaagaacagc aggaagtacc accagaaacaaatagaaaaa 961 cagatgatcc agaacaaaaa gcaaaagtta agaaaaagaa gcctaaacttttaaataaat 1021 ttgataagac tattaaagct gaacttgatg ctgcagaaaa actccgtaaaaggggaaaaa 1081 ttgaggaagc agtgaatgca tttaaagaac tagtacgcaa ataccctcagagtccacgag 1141 caagatatgg gaaggcgcag tgtgaggatg atttggctga gaagaggagaagtaatgagg 1201 tgctacgtgg agccatcgag acctaccaag aggtggccag cctacctgatgtccctgcag 1261 acctgctgaa gctgagtttg aagcgtcgct cagacaggca acaatttctaggtcatatga 1321 gaggttccct gcttaccctg cagagattag ttcaactatt tcccaatgatacttccttaa 1381 aaaatgacct tggcgtggga tacctcttga taggagataa tgacaatgcaaagaaagttt 1441 atgaagaggt gctgagtgtg acacctaatg atggctttgc taaagtccattatggcttca 1501 tcctgaaggc acagaacaaa attgctgaga gcatcccata tttaaaggaaggaatagaat 1561 ccggagatcc tggcactgat gatgggagat tttatttcca cctgggggatgccatgcaga 1621 gggttgggaa caaagaggca tataagtggt atgagcttgg gcacaagagaggacactttg 1681 catctgtctg gcaacgctca ctctacaatg tgaatggact gaaagcacagccttggtgga 1741 ccccaaaaga aacgggctac acagagttag taaagtcttt agaaagaaactggaagttaa 1801 tccgagatga aggccttgca gtgatggata aagccaaagg tctcttcctgcctgaggatg 1861 aaaacctgag ggaaaaaggg gactggagcc agttcacgct gtggcagcaaggaagaagaa 1921 atgaaaatgc ctgcaaagga gctcctaaaa cctgtacctt actagaaaagttccccgaga 1981 caacaggatg cagaagagga cagatcaaat attccatcat gcaccccgggactcacgtgt 2041 ggccgcacac agggcccaca aactgcaggc tccgaatgca cctgggcttggtgattccca 2101 aggaaggctg caagattcga tgtgccaacg agaccaggac ctgggaggaaggcaaggtgc 2161 tcatctttga tgactccttt gagcacgagg tatggcagga tgcctcatctttccggctga 2221 tattcatcgt ggatgtgtgg catccggaac tgacaccaca gcagagacgcagccttccag 2281 caatttagca tgaattcatg caagcttggg aaactctgga gaga GENBANKAccession No. S83325; codon encoding initiating methionine isunderlined).

[0015] (SEQ ID NO:3 ; GENBANK Accession No. S83325; codon encodinginitiating methionine is underlined).

[0016] Methods of inhibiting tumor growth also include administering acompound which inhibits HAAH hydroxylation of a NOTCH polypeptide. Forexample, the compound inhibits hydroxylation of an EGF-likecysteine-rich repeat sequence in a NOTCH polypeptide, e.g., onecontaining the consensus sequence CDXXXCXXKXGNGXCDXXCNNAACXXDGXDC (SEQID NO:4). Polypeptides containing an EGF-like cysteine-rich repeatsequence are administered to block hydroxylation of endogenous NOTCH.

[0017] Growth of a tumor which overexpresses HAAH is also inhibited byadministering a compound which inhibits signal transduction through theinsulin receptor substrate (IRS) signal transduction pathway. Preferablythe compound inhibits IRS phosphorylation. For example, the compound isa peptide or non-peptide compound which binds to and inhibitsphosphorylation at residues 46, 465, 551, 612, 632, 662, 732, 941, 989,or 1012 of SEQ ID NO:5. Compounds include polypeptides such those whichblock an IRS phosphorylation site such as a Glu/Tyr site. Antibodiessuch as those which bind to a carboxyterminal domain of IRS containing aphosphorylation site block IRS phosphorylation, and as a consequence,signal transduction along the pathway. Inhibition of IRS phosphorylationin turn leads to inhibition of cell proliferation. Other compounds whichinhibit IRS phosphorylation include vitamin D analogue EB1089 andWortmannin.

[0018] HAAH-overproducing tumor cells were shown to express HAAH bothintracellularly and on the surface of the tumor cell. Accordingly, amethod of killing a tumor cell is carried out by contacting such a tumorcell with a cytotoxic agent linked to an HAAH-specific antibody. TheHAAH-specific antibody (antibody fragment, or ligand which binds toextracellular HAAH) directs the chimeric polypeptide to the surface ofthe tumor cell allowing the cytotoxic agent to damage or kill the tumorcell to which the antibody is bound. The monoclonal antibody binds to anepitope of HAAH such as an epitope exposed on the surface of the cell orin the catalytic site of HAAH. The cytotoxic composition preferentiallykills tumor cells compared to non-tumor cell.

[0019] Screening methods to identify anti-tumor agents which inhibit thegrowth of tumors which overexpress HAAH are also within the invention. Ascreening method used to determine whether a candidate compound inhibitsHAAH enzymatic activity includes the following steps: (a) providing aHAAH polypeptide, e.g., a polypeptide which contains the carboxyterminalcatalytic site of HAAH; (b) providing a polypeptide comprising anEGF-like domain; (c) contacting the HAAH polypeptide or the EGF-likepolypeptide with the candidate compound; and (d) determininghydroxylation of the EGF-like polypeptide of step (b). A decrease inhydroxylation in the presence of the candidate compound compared to thatin the absence of said compound indicates that the compound inhibitsHAAH hydroxylation of EGF-like domains in proteins such as NOTCH.

[0020] Anti-tumor agents which inhibit HAAH activation of NOTCH areidentified by (a) providing a cell expressing HAAH; (b) contacting thecell with a candidate compound; and (c) measuring translocation ofactivated NOTCH to the nucleus of said cell. Translocation is measuredby using a reagent such as an antibody which binds to a 110 kDaactivation fragment of NOTCH. A decrease in translocation in thepresence of the candidate compound compared to that in the absence ofthe compound indicates that the compound inhibits HAAH activation ofNOTCH, thereby inhibiting NOTCH-mediated signal transduction andproliferation of HAAH-overexpressing tumor cells.

[0021] Nucleotide and amino acid comparisons described herein werecarried out using the Lasergene software package (DNASTAR, Inc.,Madison, Wis.). The MegAlign module used was the Clustal V method(Higgins et al., 1989, CABIOS 5(2):151-153). The parameter used were gappenalty 10, gap length penalty 10.

[0022] Hybridization is carried out using standard techniques, such asthose described in Ausubel et al. (Current Protocols in MolecularBiology, John Wiley & Sons, 1989). “High stringency” refers to nucleicacid hybridization and wash conditions characterized by high temperatureand low salt concentration, e.g., wash conditions of 65° C. at a saltconcentration of 0.1×SSC. “Low” to “moderate” stringency refers to DNAhybridization and wash conditions characterized by low temperature andhigh salt concentration, e.g., wash conditions of less than 60° C. at asalt concentration of at least 1.0×SSC. For example, high stringencyconditions include hybridization at 42° C. in the presence of 50%formamide; a first wash at 65° C. in the presence of 2×SSC and 1% SDS;followed by a second wash at 65° C. in the presence of 0.1%×SSC. Lowerstringency conditions suitable for detecting DNA sequences having about50% sequence identity to an HAAH gene sequence are detected by, forexample, hybridization at about 42° C. in the absence of formamide; afirst wash at 42° C., 6×SSC, and 1% SDS; and a second wash at 50° C.,6×SSC, and 1% SDS.

Other features and advantages of the invention will be apparent from thefollowing description of the preferred embodiments thereof, and from theclaims. BRIEF DESCRIPTION OF THE DRAWINGS

[0023]FIG. 1 is a bar graph showing colony formation induced bytransient transfection of NIH-3T3 cells with various AAH cDNAs. Colonyformation was induced by transient transfection with 10 μg DNA. Incontrast, the mutant murine AAH construct without enzymatic activity hasno transforming activity. The data is presented as mean number oftransformed foci±SEM.

[0024]FIG. 2 is a bar graph showing the results of a densitometricanalysis of a Western blot assay of proteins produced by various murineAAH stably transfected cell clones. In clones 7 and 18, there was amodest increase in HAAH gene expression, while the overexpression was toa lesser degree in clone 16.

[0025] FIGS. 3A-B are bar graphs showing colony formation in soft agarexhibited by HAAH stably transfected clones compared to HAAH enzymaticactivity. FIG. 3A shows a measurement of murine AAH enzymatic activityin clones 7, 16 and 18, and FIG. 3B shows colony formation exhibited byclones 7, 16 and 18. Data is presented as mean number of colonies 10days after plating±SEM. All three clones with modest increases in HAAHenzymatic activity, that correlated with protein expression, exhibitedanchorage independent growth.

[0026]FIG. 4 is a bar graph showing tumor formation in nude miceinjected with transfected clones overexpressing murine AAH. Tumor growthwas assessed after 30 days. Mean tumor weight observed in mice injectedwith clones 7, 16 and 18 as compared to mock DNA transfected clone. Allanimals injected with clones overexpressing HAAH developed tumors.

[0027] FIGS. 5A-D are bar graphs showing increased AAH expression inPNET2 (FIG. 5A, 5C) and SH-Sy5y (FIG. 5B) cells treated with retinoicacid (FIGS. 5A, 5B) or phorbol ester myristate (PMA; FIG. 5C) to induceneurite outgrowth as occurs during tumor cell invasion. The cells weretreated with 10 μM retinoic acid or 100 nM PMA for 0, 1, 2, 3, 4, or 7days. Cell lysates were analyzed by Western blot analysis using anHAAH-specific monoclonal antibody to detect the 85 kDa AAH protein. Thelevels of immunoreactivity were measured by volume densitometry(arbitrary units). The graphs indicate the mean±S.D. of results obtainedfrom three separate experiments. In FIG. 5D, PNET2 cells were treatedfor 24 hours with sub-lethal concentrations of H₂O₂ to induce neuriteretraction. Viability of greater than 90% of the cells was demonstratedby Trypan blue dye exclusion. Similar results were obtained for SH-Sy5ycells.

[0028]FIG. 6 is a bar graph showing the effects of AAH over-expressionon the levels of anti-apoptosis (Bcl-2), cell cycle-mitotic inhibitor(p16 and p21/Waf1), and proliferation (proliferating cell nuclearantigen; PCNA) molecules. PNET2 neuronal cells were stably transfectedwith the full-length human cDNA encoding AAH (pHAAH) or empty vector(pcDNA). AAH gene expression was under control of a CMV promoter.Western blot analysis was performed with cell lysates prepared fromcultures that were 70 to 80 percent confluent. Protein loading wasequivalent in each lane. Replicate blots were probed with the differentantibodies. Bar graphs depict the mean S.D.'s of protein expressionlevels measured in three experiments. All differences are statisticallysignificant by Student T-test analysis (P<0.01-P<0.001).

[0029]FIG. 7 is a diagram of showing the components of the IRS-1 signaltransduction pathway.

[0030]FIG. 8 is a line graph showing growth curves generated in cellsexpressing the antisense HAAH compared to controls expressing GFP.

[0031]FIG. 9 is a diagram of the functional domains of the hIRS-1protein and structural organization of the point mutants. All mutant and“wild type” hIRS-1 proteins construct contain a FLAG (F) epitope(DYKDDDDK; SEQ ID NO:7) at the C-terminus. PH and PTB indicatepleckstrin homology and phosphotyrosine binding, regions, respectively.

DETAILED DESCRIPTION

[0032] HAAH is a protein belonging to the (α-ketoglutarate dependentdioxygenase family of prolyl and lysyl hydroxylases which play a keyrole in collagen biosynthesis. This molecule hydroxylates aspartic acidor asparagine residues in EGF-like domains of several proteins in thepresence of ferrous iron. These EGF-like domains contain conservedmotifs, that form repetitive sequences in proteins such as clottingfactors, extracellular matrix proteins, LDL receptor, NOTCH homologuesor NOTCH ligand homologues.

[0033] The alpha-ketoglutarate-dependent dioxygenase aspartyl(asparaginyl) beta-hydroxylase (AAH) specifically hydroxylates oneaspartic or asparagine residue in EGF-like domains of various proteins.The 4.3-kb cDNA encoding the human AspH (hAspH) hybridizes with 2.6 kband 4.3 kb transcripts in transformed cells, and the deduced amino acidsequence of the larger transcript encodes an protein of about 85 kDa.Both in vitro transcription and translation and Western blot analysisalso demonstrate a 56-kDa protein that may result from posttranslationalcleavage of the catalytic C terminus.

[0034] An physiological function of AAH is the post-translationalbeta-hydroxylation of aspartic acid in vitamin K-dependent coagulationproteins. However, the abundant expression of AAH in several malignantneoplasms, and low levels of AAH in many normal cells indicate a rolefor this enzyme in malignancy. The AAH gene is also highly expressed incytotrophoblasts, but not syncytiotrophoblasts of the placenta.Cytotrophoblasts are invasive cells that mediate placental implantation.The increased levels of AAH expression in human cholangiocarcinomas,hepatocellular carcinomas, colon cancers, and breast carcinomas wereprimarily associated with invasive or metastatic lesions. Moreover,overexpression of AAH does not strictly reflect increased DNA synthesisand cellular proliferation since high levels of AAH immunoreactivitywere observed in 100 percent of cholangiocarcinomas, but not in human orexperimental disease processes associated with regeneration ornonneoplastic proliferation of bile ducts. AAH overexpression andattendant high levels of beta hydroxylase activity lead to invasivegrowth of transformed neoplastic cells. Detection of an increase in HAAHexpression is useful for early and reliable diagnosis of the cancertypes which have now been characterized as overexpressing this geneproduct.

[0035] Diagnosis of Malignant Tumors

[0036] HAAH is overexpressed in many tumors of endodermal origin and inat least 95% of CNS tumors compared to normal noncancerous cells. Anincrease in HAAH gene product in a patient-derived tissue sample (e.g.,solid tissue or bodily fluid) is carried out using standard methods,e.g., by Western blot assays or a quantitative assay such as ELISA. Forexample, a standard competitive ELISA format using an HAAH-specificantibody is used to quantify patient HAAH levels. Alternatively, asandwich ELISA using a first antibody as the capture antibody and asecond HAAH-specific antibody as a detection antibody is used.

[0037] Methods of detecting HAAH include contacting a component of abodily fluid with an HAAH-specific antibody bound to solid matrix, e.g.,microtiter plate, bead, dipstick. For example, the solid matrix isdipped into a patient-derived sample of a bodily fluid, washed, and thesolid matrix is contacted with a reagent to detect the presence ofimmune complexes present on the solid matrix.

[0038] Proteins in a test sample are immobilized on (bound to) a solidmatrix. Methods and means for covalently or noncovalently bindingproteins to solid matrices are known in the art. The nature of the solidsurface may vary depending upon the assay format. For assays carried outin microtiter wells, the solid surface is the wall of the well or cup.For assays using beads, the solid surface is the surface of the bead. Inassays using a dipstick (i.e., a solid body made from a porous orfibrous material such as fabric or paper) the surface is the surface ofthe material from which the dipstick is made. Examples of useful solidsupports include nitrocellulose (e.g., in membrane or microtiter wellform), polyvinyl chloride (e.g., in sheets or microtiter wells),polystyrene latex (e.g., in beads or microtiter plates, polyvinylidinefluoride (known as IMMULON™), diazotized paper, nylon membranes,activated beads, and Protein A beads. The solid support containing theantibody is typically washed after contacting it with the test sample,and prior to detection of bound immune complexes. Incubation of theantibody with the test sample is followed by detection of immunecomplexes by a detectable label. For example, the label is enzymatic,fluorescent, chemiluminescent, radioactive, or a dye. Assays whichamplify the signals from the immune complex are also known in the art,e.g., assays which utilize biotin and avidin.

[0039] An HAAH-detection reagent, e.g., an antibody, is packaged in theform of a kit, which contains one or more HAAH-specific antibodies,control formulations (positive and/or negative), and/or a detectablelabel. The assay may be in the form of a standard two-antibody sandwichassay format known in the art.

[0040] Production of HAAH-specific Antibodies

[0041] Anti-HAAH antibodies were obtained by techniques well known inthe art. Such antibodies are polyclonal or monoclonal. Polyclonalantibodies were obtained, for example, by the methods described in Ghoseet al., Methods in Enzymology, Vol. 93, 326-327, 1983. An HAAHpolypeptide, or an antigenic fragment thereof, was used as the immunogento stimulate the production of polyclonal antibodies in the antisera ofrabbits, goats, sheep, or rodents. Antigenic polypeptides for productionof both polyclonal and monoclonal antibodies useful as immunogensinclude polypeptides which contain an HAAH catalytic domain. Forexample, the immunogenic polypeptide is the full-length mature HAAHprotein or an HAAH fragment containing the carboxyterminal catalyticdomain e.g., an HAAH polypeptide containing the His motif of SEQ IDNO:2.

[0042] Antibodies which bind to the same epitopes as those antibodiesdisclosed herein as identified using standard methods, e.g., competitivebinding assays, known in the art.

[0043] Monoclonal antibodies were obtained by standard techniques. Tenμg of purified recombinant HAAH polypeptide was administered to miceintraperitoneally in complete Freund's adjuvant, followed by a singleboost intravenously (into the tail vein) 3-5 months after the initialinoculation. Antibody-producing hybridomas were made using standardmethods. To identify those hybridomas producing antibodies that arehighly specific for an HAAH polypeptide, hybridomas were screened usingthe same polypeptide immunogen used to immunize. Those antibodies whichwere identified as having HAAH-binding activity are also screened forthe ability to inhibit HAAH catalytic activity using the enzymaticassays described below. Preferably, the antibody has a binding affinityof at least about 10⁸ liters/mole and more preferably, an affinity of atleast about 10⁹ liters/mole.

[0044] Monoclonal antibodies are humanized by methods known in the art,e.g, MAbs with a desired binding specificity can be commerciallyhumanized (Scotgene, Scotland; Oxford Molecular, Palo Alto, Calf.).

[0045] HAAH-specific intrabodies are produced as follows. Followingidentification of a hybridoma producing a suitable monoclonal antibody,DNA encoding the antibody is cloned. DNA encoding a single chainHAAH-specific antibody in which heavy and light chain variable domainsare separated by a flexible linker peptide is cloned into an expressionvector using known methods (e.g., Marasco et al., 1993, Proc. Natl.Acad. Sci. USA 90:7889-7893 and Marasco et al., 1997, Gene Therapy4:11-15). Such constructs are introduced into cells, e.g., usingstandard gene delivery techniques for intracellular production of theantibodies. Intracellular antibodies, i.e., intrabodies, are used toinhibit signal transduction by HAAH. Intrabodies which bind to acarboxyterminal catalytic domain of HAAH inhibit the ability of HAAH tohydroxylate EGF-like target sequences.

[0046] Methods of linking HAAH-specific antibodies (or fragmentsthereof) which bind to cell surface exposed epitopes of HAAH on thesurface of a tumor cell are linked to known cytotoxic agents, e.g, ricinor diptheria toxin, using known methods.

[0047] Methods of Treating Malignant Tumors

[0048] Patients with tumors characterized as overexpressing HAAH as suchtumors of endodermal origin or CNS tumors are treated by administeringHAAH antisense nucleic acids.

[0049] Antisense therapy is used to inhibit expression of HAAH inpatients suffering from hepatocellular carcinomas, cholangiocarcinomas,glioblastomas and neuroblastomas. For example, an HAAH antisense strand(either RNA or DNA) is directly introduced into the cells in a form thatis capable of binding to the MRNA transcripts. Alternatively, a vectorcontaining a sequence which, which once within the target cells, istranscribed into the appropriate antisense mRNA, may be administered.Antisense nucleic acids which hybridize to target mRNA decrease orinhibit production of the polypeptide product encoded by a gene byassociating with the normally single-stranded mRNA transcript, therebyinterfering with translation and thus, expression of the protein. Forexample, DNA containing a promoter, e.g., a tissue-specific or tumorspecific promoter, is operably linked to a DNA sequence (an antisensetemplate), which is transcribed into an antisense RNA. By “operablylinked” is meant that a coding sequence and a regulatory sequence(s)(i.e., a promoter) are connected in such a way as to permit geneexpression when the appropriate molecules (e.g., transcriptionalactivator proteins) are bound to the regulatory sequence(s).

[0050] Oligonucleotides complementary to various portions of HAAH mRNAare tested in vitro for their ability to decrease production of HAAH intumor cells (e.g., using the FOCUS hepatocellular carcinoma (HCC) cellline) according to standard methods. A reduction in HAAH gene product incells contacted with the candidate antisense composition compared tocells cultured in the absence of the candidate composition is detectedusing HAAH-specific antibodies or other detection strategies. Sequenceswhich decrease production of HAAH in in vitro cell-based or cell-freeassays are then be tested in vivo in rats or mice to confirm decreasedHAAH production in animals with malignant neoplasms.

[0051] Antisense therapy is carried out by administering to a patient anantisense nucleic acid by standard vectors and/or gene delivery systems.Suitable gene delivery systems may include liposomes, receptor-mediateddelivery systems, naked DNA, and viral vectors such as herpes viruses,retroviruses, adenoviruses and adeno-associated viruses, among others. Areduction in HAAH production results in a decrease in signaltransduction via the IRS signal transduction pathway. A therapeuticnucleic acid composition is formulated in a pharmaceutically acceptablecarrier. The therapeutic composition may also include a gene deliverysystem as described above. Pharmaceutically acceptable carriers arebiologically compatible vehicles which are suitable for administrationto an animal: e.g., physiological saline. A therapeutically effectiveamount of a compound is an amount which is capable of producing amedically desirable result such as reduced production of an HAAH geneproduct or a reduction in tumor growth in a treated animal.

[0052] Parenteral administration, such as intravenous, subcutaneous,intramuscular, and intraperitoneal delivery routes, may be used todeliver nucleic acids or HAAH-inhibitory peptides or non-peptidecompounds. For treatment of CNS tumors, direct infusion intocerebrospinal fluid is useful. The blood-brain barrier may becompromised in cancer patients, allowing systemically administered drugsto pass through the barrier into the CNS. Liposome formulations oftherapeutic compounds may also facilitate passage across the blood-brainbarrier.

[0053] Dosages for any one patient depends upon many factors, includingthe patient's size, body surface area, age, the particular nucleic acidto be administered, sex, time and route of administration, generalhealth, and other drugs being administered concurrently. Dosage forintravenous administration of nucleic acids is from approximately 106 to1022 copies of the nucleic acid molecule.

[0054] Ribozyme therapy is also be used to inhibit HAAH gene expressionin cancer patients. Ribozymes bind to specific mRNA and then cut it at apredetermined cleavage point, thereby destroying the transcript. TheseRNA molecules are used to inhibit expression of the HAAH gene accordingto methods known in the art (Sullivan et al., 1994, J. Invest. Derm.103:85S-89S; Czubayko et al., 1994, J. Biol. Chem. 269:21358-21363;Mahieu et al, 1994, Blood 84:3758-65; Kobayashi et al. 1994, Cancer Res.54:1271-1275).

[0055] Methods of Identifying Compounds that Inhibit HAAH EnzymaticActivity

[0056] Aspartyl (asparaginyl) beta-hydroxylaseydroxylase (AAH) activityis measured in vitro or in vivo. For example, HAAH catalyzesposttranslational modification of βcarbon of aspartyl and asparaginylresidues of EGF-like polypeptide domains. An assay to identify compoundswhich inhibit hydroxylase activity is carried out by comparing the levelof hydroxylation in an enzymatic reaction in which the candidatecompound is present compared to a parallel reaction in the absence ofthe compound (or a predetermined control value). Standard hydroxylaseassays carried out in a testtube are known in the art, e.g., Lavaissiereet al., 1996, J. Clin. Invest. 98:1313-1323; Jia et al., 1992, J. Biol.Chem. 267:14322-14327; Wang et al., 1991, J. Biol. Chem.266:14004-14010; or Gronke et al., 1990, J. Biol. Chem. 265:8558-8565.Hydroxylase activity is also measured using carbon dioxide (¹⁴CO₂capture assay) in a 96-well microtiter plate format (Zhang et al., 1999,Anal. Biochem. 271:137-142. These assays are readily automated andsuitable for high throughput screening of candidate compounds toidentify those with hydroxylase inhibitory activity.

[0057] Candidate compound which inhibit HAAH activation of NOTCH areidentified by detecting a reduction in activated NOTCH in a cell whichexpresses or overexpresses HAAH, e.g., FOCUS HCC cells. The cells arecultured in the presence of a candidate compound. Parallel cultures areincubated in the absence of the candidate compound. To evaluate whetherthe compound inhibits HAAH activation of NOTCH, translocation ofactivated NOTCH to the nucleus of the cell is measured. Translocation ismeasured by detecting a 110 kDa activation fragment of NOTCH in thenucleus of the cell. The activation fragment is cleaved from the large(approximately 300 kDa) transmembrane NOTCH protein upon activation.Methods of measuring NOTCH translocation are known, e.g, those describedby Song et al., 1999, Proc. Natl. Acad. Sci U.S.A. 96:6959-6963 orCapobianco et al., 1997, Mol. Cell Biol. 17:6265-6273. A decrease intranslocation in the presence of the candidate compound compared to thatin the absence of the compound indicates that the compound inhibits HAAHactivation of NOTCH, thereby inhibiting NOTCH-mediated signaltransduction and proliferation of HAAH-overexpressing tumor cells.

[0058] Methods of screening for compounds which inhibit phosphorylationof IRS are carried out by incubating IRS-expressing cells in thepresence and absence of a candidate compound and evaluating the level ofIRS phosphorylation in the cells. A decrease in phosphorylation in cellscultured in the presence of the compound compared to in the absence ofthe compound indicates that the compound inhibits IRS-1 phosphorylation,and as a result, growth of HAAH-overexpressing tumors. Alternatively,such compounds are identified in an in vitro phosphorylation assay knownin the art, e.g., one which measured phosphorylation of a syntheticsubstrate such as poly (Glu/Tyr).

EXAMPLE 1

[0059] Increased Expression of HAAH is Associated with MalignantTransformation

[0060] HAAH is a highly conserved enzyme that hydroxylates EGF-likedomains in transformation associated proteins. The HAAH gene isoverexpressed in human hepatocellular carcinomas andcholangiocarcinomas. HAAH gene expression was found to be undetectableduring bile duct proliferation in both human disease and rat modelscompared to cholangiocarcinoma. Overexpression of HAAH in NIH-3T3 cellswas associated with generation of a malignant phenotype, and enzymaticactivity was found to be required for cellular transformation. The datadescribed below indicate that overexpression of HAAH is linked tocellular transformation of biliary epithelial cells.

[0061] To identify molecules that are specifically overexpressed intransformed malignant cells of human hepatocyte origin, the FOCUShepatocellular carcinoma (HCC) cell line was used as an immunogen togenerate monoclonal antibodies (mAb) that specifically or preferentiallyrecognize proteins associated with the malignant phenotype. A lambdaGT11 cDNA expression library derived from HepG2 HCC cells was screened,and HAAH-specific mAb produced against the FOCUS cell line was found torecognize an epitope on a protein encoded by an HAAH cDNA. The HAAHenzyme was found to be upregulated in several different humantransformed cell lines and tumor tissues compared to adjacent humantissue counterparts. The overexpressed HAAH enzyme in different humanmalignant tissues was found to be catalytically active.

[0062] HAAH gene expression was examined in proliferating bile ducts andin NIH 3T3 cells. Its role in the generation of the malignant phenotypewas measured by the formation of transformed foci, growth in soft agaras an index of anchorage independent growth and tumor formation in nudemice. The role of enzymatic activity in the induction of transformedphenotype was measured by using a cDNA construct with a mutation in thecatalytic site that abolished hydroxylase activity. The resultsindicated that an increase in expression of HAAH gene is associated withmalignant transformation of bile ducts.

[0063] The following materials and methods were used to generate thedata described below.

[0064] Antibodies

[0065] The FB50 monoclonal antibody was generated by cellularimmunization of Balb/C mice with FOCUS HCC cells. A monoclonalanti-Dengue virus antibody was used as a non-relevant control. The HBOH2monoclonal antibody was generated against a 52 kDa recombinant HAAHpolypeptide and recognizes the catalytic domain of beta-hydroxylase frommouse and human proteins. Polyclonal anti-HAAH antibodies cross-reactwith rat hydroxylase protein. Control antibody anti-Erk-1 was purchasedfrom Santa Cruz Biotechnology, Inc, CA. Sheep anti-mouse and donkeyanti-rabbit antisera labeled with horseradish peroxidase were obtainedfrom Amersham, Arlington Heights, Ill.

[0066] Constructs

[0067] The murine full length AAH construct (pNH376) and thesite-directed mutation construct (pNH376-H660) with abolished catalyticactivity were cloned into the eukaryotic expression vector pcDNA3(Invitrogen Corp., San Diego, Calif.). The full length human AAH wascloned into prokaryotic expression vector PBC-SK+ (Stratagene, La Jolla,Calif.). The full length human AAH (GENBANK Accession No. S83325) wassubcloned into the EcoRI site of the pcDNA3 vector.

[0068] Animal Model of Bile Duct Proliferation

[0069] Rats were divided into 9 separate groups of 3 animals each exceptfor group 9 which contained 5 rats. Group 1 was the non-surgical controlgroup, and group 2 was the sham-operated surgical control. The remaininggroups underwent common bile duct ligation to induce intrahepatic bileduct proliferation and were evaluated at 6, 12, 24, 48 hours and 4, 8and 16 days as shown in Table 3. Animals were asphyxiated with CO₂, andliver samples were taken from left lateral and median lobes, fixed in 2%paraformaldehyde and embedded in paraffin. Liver samples (5 μm) were cutand stained with hematoxylin and eosin to evaluate intrahepatic bileduct proliferation. Immunohistochemistry was performed with polyclonalanti-HAAH antibodies that cross-react with the rat protein to determinelevels of protein expression.

[0070] Bile Duct Proliferation Associated with Primary SclerosingCholangitis (PSC)

[0071] Liver biopsy samples were obtained from 7 individuals with PSCand associated bile duct proliferation. These individuals were evaluatedaccording to standard gastroenterohepatological protocols. Patients were22-46 years of age and consisted of 4 males and 3 females. Four hadassociated inflammatory bowel disease (3 ulcerative colitis and 1Crohn's colitis). All patients underwent a radiological evaluationincluding abdominal ultrasonography and endoscopic retrogradecholangiopancreaticography to exclude the diagnosis of extrahepaticbiliary obstruction. Tissue sections were prepared from paraffinembedded blocks and were evaluated by hematoxylin and eosin staining forbile duct proliferation. Expression of HAAH was determined byimmunohistochemistry using an HAAH-specific monoclonal antibody such asFB50.

[0072] Immunohistochemistry

[0073] Liver tissue sections (5 μm) were deparaffinized in xylene andrehydrated in graded alcohol. Endogenous peroxidase activity wasquenched by a 30-minute treatment with 0.6% H₂O₂ in 60% methanol.Endogenous biotin was masked by incubation with avidin-biotin blockingsolutions (Vector Laboratories, Burlingame, Calif.). The FB50 mAb (forPSC samples) and polyclonal anti-HAAH-hydroxylase antibodies (for ratliver samples) were added to slides in a humidified chamber at 4° C.overnight. Immunohistochemical staining was performed using a standardavidin-biotin horseradish peroxidase complex (ABC) method usingVectastain Kits with diaminobenzidine (DAB) as the chromogen accordingto manufacturer's instructions (Vector Laboratories, Inc., Burlingame,Calif.). Tissue sections were counterstained with hematoxylin, followedby dehydration in ethanol. Sections were examined by a light microscopyfor bile duct proliferation and HAAH protein expression. Paraffinsections of cholangiocarcinoma and placenta were used as positivecontrols, and hepatosteatosis samples were used as a negative controls.To control for antibody binding specificity, adjacent sections wereimmunostained in the absence of a primary antibody, or usingnon-relevant antibody to Dengue virus. As a positive control for tissueimmunoreactivity, adjacent sections of all specimens were immunostainedwith monoclonal antibody to glyceraldehyde 3-phosphate dehydrogenase.

[0074] Western Blot Analysis

[0075] Cell lysates were prepared in a standard radioimmunoprecipitationassay (RIPA) buffer containing protease inhibitors. The total amount ofprotein in the lysates was determined by Bio-Rad calorimetric assay (BioRad, Hercules, Calif.) followed by 10% sodium dodecylsulphate-polyacrylamide gel electrophoresis (SDS-PAGE), transferred toPVDF membranes, and subjected to Western blot analysis using FB50,HBOH2, anti-Erk-1 (used as an internal control for protein loading) asprimary, sheep anti-mouse and donkey anti-rabbit antisera labeled withhorseradish peroxidase as secondary antibodies. Antibody binding wasdetected with enhanced chemiluminescence reagents (SuperSignal, PierceChemical Company, Rockford, Ill.) and film autoradiography. The levelsof immunoreactivity were measured by volume densitometry using NIH Imagesoftware.

[0076] Enzymatic Activity Assay

[0077] AAH activity was measured in cell lysates using the firstEGF-like domain of bovine protein S as substrate where ¹⁴C-labeledα-ketogluterate hydroxylates the domain releasing ¹⁴C containing CO2according to standard methods, e.g., those described by Jia et al.,1992, J. Biol. Chem. 267:14322-14327; Wang et al., 1991, J. Biol. Chem.266:14004-14010; or Gronke et al., 1990, J. Biol. Chem. 265:8558-8565.Incubations were carried out at 37° C. for 30 min in a final volume of40 μl containing 48 μg of crude cell extract protein and 75 μM EGFsubstrate.

[0078] Cell Transfection Studies

[0079] The NIH-3T3 cells were cultured in Dulbecco's modified Eagle'smedium (DMEM; Mediatech, Washington, D.C.) supplemented with 10%heat-inactivated fetal calf serum (FCS; Sigma Chemical Co., St.Louis,Mo.), 1% L-glutamine, 1% non-essential amino acids and 1%penicillin-streptomycin (GIBCO BRL, Life Technologies, Inc., GrandIsland, N.Y.). Subconfluent NIH-3T3 cells (3×10⁵ cells/60-mm dish) weretransfected with 10 μg of one of the following plasmids: 1)non-recombinant pcDNA3 vector (Invitrogen Corp., San Diego, Calif.) as anegative control; 2) pNH376-H660, the murine AAH cDNA that was mutatedin the catalytic domain and cloned into the pcDNA3 vector driven by aCMV promoter; 3) pNH376, the wild type murine AAH cDNA cloned into thepcDNA3 vector; 4) pCDHH, wild type human AAH cDNA cloned into the pcDNA3vector; or 5) pLNCX-UP1, a cDNA that encodes v-Src oncogene (positivecontrol). Cells were transfected using the calcium phosphatetransfection kit according to manufacturer's instructions (5 Prime—3Prime, Inc., Boulder, Colo.). Comparison of cellular transfectionefficiency was assessed with the various constructs. For this procedure,confluent plates obtained 48 hours after transfection were split andreseeded into 12 separate 6-cm dishes, and 6 of them were made to growin the presence of 400 μg/ml G-418 (GIBCO BRL, Life Technologies, Inc.,Grant Island, N.Y.) containing medium. The number of G-418 resistantfoci was determined at 14 days after transfection and used to correctfor any variability in transfection efficiency.

[0080] Transformation Assay

[0081] The NIH-3T3 cells were transfected with the various constructsand allowed to reach confluence after 48 hours as described above. Each6 cm dish was split and seeded into 12 different 6 cm dishes. While 6 ofthem were made to grow in the presence of G-418 to detect transfectionefficiency, the other six were grown in complete medium without G-418and with a medium change every 4th day. The number of transformed fociwere counted in these plates without G-418 and expressed as transformedfoci per μg transfected DNA.

[0082] Anchorage-independent Cell Growth Assay

[0083] A limiting dilution technique (0.15 cell/well of a flat bottom96-well-plate) was performed on transfectants grown in G-418 in order toisolate cell clones with different levels of HAAH activity as measuredby Western blot analysis and enzymatic assay of hydroxylase activity.Cloned cell lines (1.0×10⁴ cells) were suspended in complete mediumcontaining 0.4% low-melting agarose (SeaPlaque GTG Agarose; FMCBioproducts, Rockland, Me.) and laid over a bottom agar mixtureconsisting of complete medium with 0.53% low-melting agarose. Each clonewas assayed in triplicate. The clones were seeded under these conditionsand 10 days later the size (positive growth>0.1 mm in diameter) andnumber of foci were determined.

[0084] Tumorigenicity in Nude Mice

[0085] The same clones as assessed in the anchorage independent growthassay were injected into nude mice and observed for tumor formation.Tumorigenicity was evaluated using 10 animals in each of 4 groups(Charles River Labs., Wilmington, Mass.). Group 1 received 1×10⁷ cellsstably transfected with mock DNA, Group 2-4 received 1×10⁷ cells ofclones stable transfected with pNH376 and expressing various levels ofmurine HAAH protein. Nude mice were kept under pathogen-free conditionsin a standard animal facility. Thirty days after tumor cell inoculation,the animals were sacrificed using isofluorane (Aerrane, Anaquest, N.J.)containing chambers and the tumors were carefully removed and weightdetermined.

[0086] Animal Model of Bile Duct Proliferation

[0087] Following ligation of the common bile duct, intrahepatic bileduct proliferation was evident at 48 hours. Tissue samples obtained 8and 16 days following common bile duct ligation revealed extensive bileduct proliferation as shown in Table 3. TABLE 3 Bile duct proliferationand HAAH expression at different intervals after common bile ductligation Surgical Immunohisto- Group Procedure Microscopy* chemistry 1no surgery normal negative 2 sham surgery normal negative 3 6 hours postnormal negative ligation 4 12 hours post normal negative ligation 5 24hours post normal negative ligation 6 48 hours post minimal bilenegative ligation duct prolif. 7 4 days post moderate bile negativeligation duct prolif. 8 8 days post extensive negative ligation bileduct prolif. 9 16 days post extensive negative ligation bile ductprolif.

[0088] Immunohistochemical staining failed to detect presence of HAAH inproliferating bile ducts at any time. Analysis of HAAH expression inbile ducts derived from sham surgical controls was also negative, whileall samples exhibited positive immunoreactivity with control antibodiesto glyceraldehyde 3-phosphate dehydrogenase. Thus, bile ductproliferation was not associated with increased HAAH expression in thisstandard animal model system.

[0089] HAAH Expression in PSC

[0090] The liver biopsy specimens from patients with PSC exhibited bileduct proliferation accompanied by periductal fibrosis and a mononuclearinflammatory cell infiltrate without evidence of dysplasia. Adjacentsections immunostained with the an HAAH-specific monoclonal antibody hadno detectable HAAH immunoreactivity in proliferating bile ducts. Incontrast, sections of cholangiocarcinoma that were immunostainedsimultaneously using the same antibody and detection reagents manifestedintense levels of HAAH immunoreactivity in nearly all tumor cells,whereas adjacent sections of the cholangiocarcinomas exhibited anegative immunostaining reaction with monoclonal antibody to Denguevirus. These findings indicate that HAAH expression was associated withmalignant transformation rather than non-cancerous cellularproliferation of intrahepatic bile ducts.

[0091] HAAH Associated Transformation of NIH-3T3 Cells

[0092] The transforming capability of the murine and human AAH genes, aswell as the murine AAH mutant construct without enzymatic activity werecompared to mock DNA (negative control) and v-Src transfected NIH-3T3cells (positive control). The transforming capability of murine AAH wasfound to be 2-3 times that of vector DNA control as shown in FIG. 1. Thetransforming capacity of the human gene was greater than that observedwith the murine AAH (32±1.5 versus 13±2.6 transformed foci,respectively). The murine and human AAH transfected cells formed largefoci, resembling those of v-Src transfected fibroblasts, compared to theoccasional much smaller foci observed in cells transfected with vectorDNA that displayed the contact inhibition of fibroblast cell lines.Parallel experiments performed using the mutant pNH376-H660 constructwithout enzymatic activity revealed no transforming activity. Thisfinding indicates that the enzymatic activity of HAAH is required forthe transforming activity exhibited by the HAAH gene.

[0093] Anchorage-independent Cell Growth Assay

[0094] After transient transfection with the murine AAH construct,several different transformed foci were isolated for dilutional cloningexperiments to establish stable transfected cell clones with differentlevels of HAAH gene expression. Nine different cloned cell lines wereselected for further study. The expression level of the HAAH protein wasdetermined by Western blot analysis. Clones 7 and 18 had a modestincrease in HAAH protein expression, yet formed large colonies in softagar (FIG. 2). Protein loading was equivalent in all lanes as shown byimmunoblotting of the same membranes with an anti-Erk-1 monoclonalantibody. The increased protein expression was associated with increasedenzymatic activity as shown in FIG. 3. The capability of these clones toexhibit anchorage independent cell growth in soft agar is presented inFIG. 3. All 3 clones with increased HAAH gene expression demonstratedanchorage independent cell growth compared to the mock DNA transfectedclone.

[0095] Tumor Formation in Nude Mice

[0096] The 3 clones with increased HAAH gene expression were evaluatedfor the ability to form tumors in nude mice. Tumor size in the mousegiven clone 18 was compared to a mock DNA transfected clone. Clones 7,16 and 18 were highly transformed in this assay and produced largetumors with a mean weight of 2.5, 0.9 and 1.5 grams, respectively (FIG.4). These data indicate that overexpression of HAAH contributes toinduction and maintenance of the malignant phenotype in vivo.

[0097] High Level HAAH Expression is Indicative of Malignancy

[0098] In order to determine if HAAH expression was associated withmalignancy rather than increased cell turnover, two models of bile ductproliferation were studied. In the animal model, ligation of the commonbile duct induced extensive intrahepatic bile duct proliferation, yetthere was no evidence of HAAH gene expression under these experimentalconditions as shown in Table 3. Similarly, HAAH gene expression wasassessed in a human disease model associated with bile ductproliferation since PSC is an autoimmune liver disease associated withdestruction as well as proliferation of the intra and extrahepatic bileducts. PSC is premalignant disease, and a significant proportion ofaffected individuals will eventually develop cholangiocarcinoma.However, no evidence for increased HAAH gene expression in the presenceof extensive bile duct proliferation.

[0099] Having established that HAAH protein levels were elevated incholangiocarcinoma and not in normal or proliferating bile ducts, therole of HAAH in the generation of a malignant phenotype was studied. TheHAAH gene was transfected into NIH-3T3 cells and cellular changes, e.g.,increased formation of transformed foci, colony growth in soft agar andtumor formation in nude mice associated with malignant transformation,were evaluated. The full-length murine and human AAH genes were clonedinto expression constructs and transiently transfected into NIH-3T3cells. An increased number of transformed foci was detected in cellstransfected both with the murine and human AAH genes as compared to mockDNA transfected controls. The increased number of transformed foci,after controlling for transfection efficiency, was not as high comparedto v-Src gene transfected cells used as a positive control. Theenzymatic activity of the HAAH gene was required for a malignantphenotype because a mutant construct which abolished the catalytic sitehad no transforming properties. Several stable transfectants and clonedNIH-3T3 cell lines with a modest increase in HAAH protein levels andenzymatic activity were established. Such cell lines were placed in softagar to examine anchorage independent cell growth as another property ofthe malignant phenotype. All cell lines grew in soft agar compared tomock DNA transfected control, and there was a positive correlationbetween the cellular level of HAAH gene expression and the number andsize of colonies formed. Three of these cloned cell lines formed tumorsin nude mice. All three cell lines with increased HAAH expression wereoncogenic as shown by the development of large tumors as anotherwell-known characteristic of the transformed phenotype.

[0100] To determine whether cellular changes induced by overexpressionof HAAH were related to the enzymatic function, a site-directed mutationwas introduced into the gene that changed the ferrous iron binding sitefrom histidine to lysine at 660th position of mouse HAAH therebyabolishing hydroxylase activity of the murine HAAH. A correspondingmutation in HAAH is used as a dominant negative mutant to inhibit HAAHhydroxylase activity. The pNH376-H660 construct had no transformationactivity indicating cellular changes of the malignant phenotype inducedby overexpression depends on the enzymatic activity of the protein.

[0101] Notch receptors and their ligands have several EGF-like domainsin the N-terminal region that contain the putative consensus sequencefor beta-hydroxylation. Notch ligands are important elements of theNotch signal transduction pathway and interaction of Notch with itsligands occurs by means of EGF-like domains of both molecules. Pointmutations affecting aspartic acid or asparagine residues in EGF-likedomains that are the targets for beta-hydroxylation by HAAH reducecalcium binding and protein-protein interactions involved in theactivation of downstream signal transduction pathways. Overexpression ofHAAH and Notch protein hydroxylation by HAAH contributes to malignancy.Tumor growth is inhibited by decreasing Notch protein hydroxylation byHAAH.

[0102] The data presented herein is evidence that high-level HAAHexpression is linked to malignant transformation. An increase inexpression of the HAAH cDNA in NIH-3T3 cells induced a transformedphenotype manifested by increased numbers of transformed foci,anchorage-independent growth, and tumorigenesis in nude mice. Inaddition, intact HAAH-enzyme was found to be required forHAAH-associated transformation. Accordingly, inhibition of as little as20% of endogenous HAAH enzymatic activity or expression confers atherapeutic benefit. For example, clinical benefit is achieved by50%-70% inhibtion of HAAH expression or activity after administarationof an HAAH inhibitory compound compared to the level associated withuntreated cancer cell or a normal noncancerous cell.

[0103] HAAH is regulated at the level of transcription. Only modestincreases in HAAH expression and enzyme activity were required forcellular transformation. These results indicate that increased HAAH geneexpression and enzyme activity contribute to the generation ormaintenance of the transformed phenotype and that decreasingtranscription of the HAAH gene or decreasing enzymatic activity of theHAAH gene product leads to a decrease in malignancy. Accordingly, HAAHtranscription is inhibited by administering compounds which decreasebinding of Fos and/or Jun (elements which regulate HAAH transcription)to HAAH promoter sequences.

[0104] Since HAAH is up-regulated with malignant transformation of bileduct epithelium, and HAAH immunoreactivity is detectable on tumor cellsurface membranes, HAAH is also a molecule to which to target acytotoxic agent, e.g., by linking the cytotoxic agent to a compound thatbinds to HAAH expressed on the surface of a tumor cell. Assay of HAAHprotein levels in either biological fluids such as bile, or cellsobtained by fine needle aspiration is a diagnostic marker of humancholangiocarcinoma.

EXAMPLE 2

[0105] Expression of AAH and Growth and Invasiveness of Malignant CNSNeoplasms

[0106] AAH is abundantly expressed in carcinomas and trophoblasticcells, but not in most normal cells, including those of CNS origin. Highlevels of AAH expression were observed in 15 of 16 glioblastomas, 8 of 9anaplastic oligodendrogliomas, and 12 of 12 primitive neuroectodermaltumors (PNETs). High levels of AAH immunoreactivity were primarilylocalized at the infiltrating edges rather than in the central portionsof tumors. Double-label immunohistochemical staining demonstrated areciprocal relationship between AAH and tenascin, a substrate for AAHenzyme activity. PNET2 neuronal cell lines treated with phorbol estermyristate or retinoic acid to stimulate neuritic extension and invasivegrowth exhibited high levels of AAH expression, whereas H₂O₂-inducedneurite retraction resulted in down-regulation of AAH. PNET2 neuronalcells that stably over-expressed the human AAH cDNA had increased levelsof PCNA and Bcl-2, and reduced levels of p21/Waf1 and p16, suggestingthat AAH overexpression results in enhanced pathological cellproliferation, cell cycle progression, and resistance to apoptosis. Inaddition, the reduced levels of p16 observed in AAH-transfectantsindicate that AAH over-expression confers enhanced invasive growth ofneoplastic cells since deletion or down-regulation of the p16 genecorrelates with more aggressive and invasive in vivo growth ofglioblastomas. Increased AAH immunoreactivity was detected at theinfiltrating margins of primary malignant CNS neoplasms, furtherindicating a role of HAAH in tumor invasiveness.

[0107] The following materials and methods were used to generate thedata described below.

[0108] Analysis of AAH Immunoreactivity in Primary Human Malignant CNSNeoplasms

[0109] AAH immunoreactivity was examined in surgical resection specimensof glioblastoma (N=16), anaplastic oligodendroglioma (N=9), andprimitive neuroectodermal tumor (PNET; supratentorial neuroblastomas(N=3) and medulloblastomas (N=9). The histopathological sections werereviewed to confirm the diagnoses using standard criteria. Paraffinsections from blocks that contained representative samples of viablesolid tumor, or tumor with adjacent intact tissue were studied. Sectionsfrom normal adult postmortem brains (N=4) were included as negativecontrols. AAH immunoreactivity was detected using qn HAAH-specificmonoclonal antibody. Immunoreactivity was revealed by the avidin-biotinhorseradish peroxidase complex method (Vector ABC Elite Kit; VectorLaboratories, Burlingame, Calif.) using 3-3′ diaminobenzidine (DAB) asthe chromogen (24) and hematoxylin as a counterstain.

[0110] Tenascin and laminin are likely substrates for AAH due to thepresence of EGF-like repeats within the molecules. Double-immunostainingstudies were performed to co-localize AAH with tenascin or laminin. TheAAH immunoreactivity was detected by the ABC method with DAB as thechromogen, and tenascin or laminin immunoreactivity was detected by theavidin-biotin alkaline phosphatase complex method (Vector Laboratories,Burlingame, Calif.) with BCIP/NBT as the substrate. As positive andnegative controls, adjacent sections were immunostained with monoclonalantibody to glial fibrillary acidic protein (GFAP) and Hepatitis Bsurface antigen. All specimens were batch immunostained using the sameantibody dilutions and immunodetection reagents.

[0111] Cell Lines and Culture Conditions

[0112] Studies were conducted to determine whether AAH expression wasmodulated with neurite (filopodia) extension (sprouting) as occurs withinvasive growth of malignant neoplasms. Human PNET2 CNS-derived andSH-Sy5y neuroblastoma cells were cultured and stimulated for 0, 1, 2, 3,5, or 7 days with 100 nM phorbol 12-ester 13-acetate or 10 μM retinoicacid to induce sprouting. In addition, to examine the effects of neuriteretraction on AAH expression, subconfluent cultures were treated for 24hours with low concentrations (10-40 μM) of H₂O₂. For both studies, AAHexpression was evaluated by Western blot analysis using the anHAAH-specific antibody.

[0113] Generation of PNET2 AAH-transfected Clones

[0114] The full-length human AAH cDNA (SEQ ID NO:3) was ligated into thepcDNA3.1 mammalian expression vector in which gene expression was underthe control of a CMV promoter (Invitrogen Corp., San Diego, Calif.).PNET2 cells were transfected with either pHAAH or pcDNA3 (negativecontrol) using Cellfectin reagent (Gibco BRL, Grand Island, N.Y.).Neomycin-resistant clones were selected for study if the constitutivelevels of AAH protein expression were increased by at least two-foldrelative to control (pcDNA3) as detected by Western blot analysis. Todetermine how AAH overexpression altered the expression of genes thatmodulate the transformed phenotype, the levels of proliferating cellnuclear antigen (PCNA), p53, p21/Waf1, Bcl-2, and p16 were measured incell lysates prepared from subconfluent cultures of AAH (N=5) and pcDNA3(N=5) stably transfected clones. PCNA was used as marker of cellproliferation. p53, p21/Waf1, and Bcl-2 levels were examined todetermine whether cells that over-expressed AAH were more prone to cellcycle progression and more resistant to apoptosis. The levels of p16were assessed to determine whether AAH over-expression has a role intumor invasiveness.

[0115] Western Blot Analysis

[0116] Cells grown in 10 cm² dishes were lysed and homogenized in astandard radioimmunoprecipitation assay RIPA buffer containing proteaseand phosphatase inhibitors. The supernatants collected aftercentrifuging the samples at 12,000×g for 10 minutes to remove insolubledebris were used for Western blot analysis. Protein concentration wasmeasured using the BCA assay (Pierce Chemical Co, Rockford, Ill.).Samples containing 60 μg of protein were electrophoresed in sodiumdodecyl sulfate polyacrylamide gels (SDS-PAGE) and subjected to Westernblot analysis. Replicate blots were probed with the individualantibodies. Immunoreactivity was detected with horseradish peroxidaseconjugated IgG (Pierce Chemical Co, Rockford, Ill.) and enhancedchemiluminescence reagents. To quantify the levels of proteinexpression, non-saturated autoradiographs were subjected to volumedensitometry using NIH Image software, version 1.6. Statisticalcomparisons between pHAAH and pcDNA3 transfected cells were made usingStudent T tests.

[0117] Antibodies

[0118] HAAH-specific monoclonal antibody generated against the FOCUShepatocellular carcinoma cells were used to detect AAH immunoreactivity.Monoclonal antibodies to tenascin, and glial fibrillary acidic protein,and rabbit polyclonal antibody to laminin were purchased from Sigma Co(St. Louis, Mo.). Rabbit polyclonal antibody to human p16 was purchasedfrom Santa Cruz Biotechnology Inc. (Santa Cruz, Calif.). The 5C3negative control monoclonal antibody to Hepatitis B surface antigen wasgenerated using recombinant protein and used as a negative control.

[0119] AAH Immunoreactivity in Primary Malignant Brains Tumors

[0120] AAH immunoreactivity was detected in 15 of 16 glioblastomas, 8 of9 anaplastic oligodendrogliomas, and all 12 PNETs. AAH immunoreactivitywas localized in the cytoplasm, nucleus, and cell processes. The tissuedistribution of AAH immunoreactivity was notable for the intenselabeling localized at the interfaces between tumor and intact brain, andthe conspicuously lower levels of immunoreactivity within the centralportions of the tumors. High levels of AAH immunoreactivity were alsoobserved in neoplastic cells distributed in the subpial zones,leptomeninges, Virchow-Robin perivascular spaces, and in individual orsmall clusters of neoplastic cells that infiltrated the parenchyma. Incontrast, AAH immunoreactivity was not detectable in normal brain. Thedistribution of AAH immunoreactivity appeared not to be strictlycorrelated with DNA synthesis since the density of nuclei in mitosis(1-5%) was similar in the central and peripheral portions of the tumors.

[0121] Relationship Between AAH and Tenascin Immunoreactivity inGlioblastomas

[0122] Tenascin is an extracellular matrix-associated antigen expressedin malignant gliomas. Tenascin contains EGF-like domains within themolecule, a substrate for HAAH hydroxylation. To localize AAH inrelation to tenascin immunoreactivity in malignant brain tumors,double-label immunohistochemical staining was performed in which AAH wasdetected using a brown chromogen (DAB), and tenascin, a blue chromogen(BCIP/NBT). Adjacent sections were similarly double-labeled toco-localize AAH with laminin, another EGF domain containingextracellular matrix molecule expressed in the CNS. Intense levels oftenascin immunoreactivity were observed in perivascular connectivetissue and in association with glomeruloid proliferation of endothelialcells. The double-labeling studies demonstrated a reciprocalrelationship between AAH and tenascin immunoreactivity such that highlevels of AAH were associated with low or undetectable tenascin, and lowlevels of AAH were associated with abundant tenascin immunoreactivity.Although laminins are also likely substrates for AAH enzyme activity dueto the EGF repeats within the molecules, double labeling studiesrevealed only low levels of laminin immunoreactivity throughout thetumors and at interfaces between tumor and intact tissue.

[0123] Analysis of AAH Expression in Neuronal Cell Lines Treated withPMA or RA

[0124] Neuritic sprouting/filopodia extension marks invasive growth ofneoplastic neuronal cells. PMA activates protein kinase C signaltransduction pathways that are involved in neuritic sprouting. Retinoicacid binds to its own receptor and the ligand-receptor complextranslocates to the nucleus where it binds to specific consensussequences present in the promoter/enhancer regions of target genesinvolved in neuritic growth. Both PNET2 and SH-Sy5y cells can be inducedto sprout by treatment with PMA (60-120 nM) or retinoic acid (5-10 μM).FIGS. 5A-D depict data from representative western blot autoradiographs;the bar graphs correspond to the mean+S.D. of results obtained fromthree experiments. Western blot analysis with the FB50 antibody detecteddoublet bands corresponding to protein with an molecular mass ofapproximately 85 kDa. Untreated PNET2 cells had relatively low levels ofAAH immunoreactivity (FIG. 5A), whereas untreated SH-Syγy cells hadreadily detected AAH expression (FIG. 5B). Untreated PNET2 cellsexhibited polygonal morphology with coarse, short radial cell processes,whereas SH-Syγy cells were slightly elongated and spontaneously extendfine tapered processes. Both cell lines manifested time-dependentincreases in the levels of AAH immunoreactivity following either RA(FIGS. 5A and 5B) or PMA (FIG. 5C) stimulation and neurite extension. InPNET2 cells, the levels of AAH protein increased by at least two-fold 24hours after exposure to RA or PMA, and high levels of AAH were sustainedthroughout the 7 days of study. In SH-Syγy cells, the RA- orPMA-stimulated increases in AAH expression occurred more gradually andwere highest after 7 days of treatment (FIG. 5B).

[0125] To examine the effect of AAH expression on neurite retraction,PNET2 and SH-Sy5y cells were treated with low concentrations (8-40 μM)of H₂O₂. After 24 hours exposure to up to 40 μM H₂O₂, although mostcells remained viable (Trypan blue dye exclusion), they exhibitedneurite retraction and rounding. Western blot analysis using the FB50antibody demonstrated H202 dose-dependent reductions in the levels ofAAH protein (FIG. 5D).

[0126] Effects of AAH Over-expression in PNET2 Cells

[0127] To directly assess the role of AAH overexpression in relation tothe malignant phenotype, PNET2 cells were stably transfected with thehuman full-length cDNA with gene expression under control of a CMVpromoter (pHAAH). Neomycin-resistant clones that had at least two-foldhigher levels of AAH immunoreactivity relative to neomycin-resistantpcDNA3 (mock) clones were studied. Since aggressive behavior ofmalignant neoplasms is associated with increased DNA synthesis, cellcycle progression, resistance to apoptosis, and invasive growth, thechanges in phenotype associated with constitutive over-expression of AAHwere characterized in relation to PCNA, p21/Waf1, p53, Bcl-2, and p16.PCNA was used as an index of DNA synthesis and cell proliferation.p21/Waf1 is a cell cycle inhibitor. Expression of the p53tumor-suppressor gene increases prior to apoptosis, whereas bcl-2inhibits apoptosis and enhances survival of neuronal cells. p16 is anoncosuppressor gene that is often either down-regulated or mutated ininfiltrating malignant neoplasms.

[0128] Five pHAAH and 5 pcDNA3 clones were studied. Increased levels ofAAH expression in the PHAAH transfected clones was confirmed by Western(FIG. 6) and Northern blot analyses. Western blot analysis using celllysates from cultures that were 70 to 80 percent confluent demonstratedthat constitutively increased levels of AAH expression (approximately 85kDa; P<0.05) in pHAAH-transfected cells were associated withsignificantly increased levels of PCNA (approximately 35 kDa; P<0.01)and Bcl-2 (approximately 25 kDa; P<0.05), and reduced levels of p21/Waf1(approximately 21 kDa; P<0.001) and p16 (approximately 16 kDa; P<0.001)(FIG. 6). However, the pHAAH stable transfectants also exhibited higherlevels of wild-type p53 (approximately 53-55 kDa). Although AAHexpression (85 kDa protein) in the stable transfectants was increased byonly 75 to 100 percent, the levels of p16 and p21/Waf1 were sharplyreduced, and PCNA increased by nearly two-fold (FIG. 6).

[0129] Increased AAH Expression is Indicative of Growth and Invasivenessof Malignant CNS Neoplasms

[0130] The data described herein demonstrates that AAH overexpression isa diagnostic tool by which to identify primary malignant CNS neoplasmsof both neuronal and glial cell origin. Immunohistochemical stainingstudies demonstrated that AAH overexpression was detectable mainly atthe interfaces between solid tumor and normal tissue, and ininfiltrating neoplastic cells distributed in the subpial zones,leptomeninges, perivascular spaces, and parenchyma. In vitro experimentsdemonstrated that AAH gene expression was modulated with neurite(filopodium) extension and invasiveness and down-regulated with neuriteretraction. In addition, PNET2 cells stably transfected with the AAHcDNA exhibited increased PCNA and bcl-2, and reduced Waf1/p21 and p16expression. Therefore, AAH overexpression contributes to the transformedphenotype of CNS cells by modulating the expression of other genes thatpromote cellular proliferation and cell cycle progression, inhibitapoptosis, or enhance tumor cell invasiveness.

[0131] The data demonstrated readily detectable AAH mRNA transcripts(4.3 kB and 2.6 kB) and proteins (85 kDa and 50-56 kDa) in PNET2 andSH-Sy5y cells, but not in normal brain. Correspondingly, high levels ofAAH immunoreactivity were observed in 35 of the 37 in malignant primaryCNS-derived neoplasms studied, whereas the 4 normal control brains hadno detectable AAH immunoreactivity. The presence of high-level AAHimmunoreactivity at the infiltrating margins and generally not in thecentral portions of the tumors indicates that AAH overexpression isinvolved in the invasive growth of CNS neoplasms. Administration ofcompounds which decrease AAH expression or enzymatic activity inhibitsproliferation of CNS tumors which overexpress AAH, as well as metastasesof CNS tumors to other tissue types.

[0132] The AAH enzyme hydroxylates EGF domains of a number of proteins.Tenascin, an extracellular matrix molecule that is abundantly expressedin malignant gliomas, contains EGF-like domains. Since tenascin promotestumor cell invasion, its abundant expression in glioblastomas representsan autocrine mechanism of enhanced tumor cell growth vis-α-vis thefrequent overexpression of EGF or EGF-like receptors in malignant glialcell neoplasms. Analysis of the functional domains of tenascinsindicated that the mitogenic effects of this family of molecules arelargely mediated by the fibronectin domains, and that the EGF-likedomains inhibit growth, cell process elongation, and matrix invasion.Therefore, hydroxylation of the EGF-like domains by AAH represents animportant regulatory factor in tumor cell invasiveness.

[0133] Double-label immunohistochemical staining studies demonstrated areciprocal relationship between AAH and tenascin immunoreactivity suchthat high levels AAH immunoreactivity present at the margins of tumorswere associated with low levels of tenascin, and low levels of AAH wereoften associated with high levels of tenascin. These observationsindicated that AAH hydroxylation of EGF-like domains of tenascin altersthe immunoreactivity of tenascin protein, and in so doing, facilitatesthe invasive growth of malignant CNS neoplasms into adjacent normaltissue and perivascular spaces.

[0134] AAH immunoreactivity was examined in PNET2 and SH-Sy5y neuronalcells induced to undergo neurite extension with PMA or retinoic acid, orneurite retraction by exposure to low doses of H₂O₂. AAH expression wassharply increased by PMA- or retinoic acid-induced neurite (filopodium)extension, and inhibited by H202-induced neurite retraction and cellrounding. Neurite or filopodium extension and attachment toextracellular matrix are required for tumor cell invasion in the CNS.The EGF-like domains of tenascin inhibit neuritic and glial cell growthinto the matrix during development.

[0135] To directly examine the role of AAH overexpression in relation tothe transformed phenotype, genes modulated with DNA synthesis, cellcycle progression, apoptosis, and tumor invasiveness were examined inneuronal cell clones that stably over-expressed the human AAH cDNA. Thefindings of increased PCNA and reduced Waf1/p21 immunoreactivityindicated that AAH overexpression enhances cellular proliferation andcell cycle progression. In addition, the finding of increased Bcl-2expression indicated that AAH overexpression contributes to thetransformed phenotype by increasing cellular resistance to apoptosis.The apparently contradictory finding of higher levels of p53 in thecells that overexpressed AAH is explained by the observation that highlevels of wildtype p53 in immature neuronal cells were associated withneuritic growth (invasiveness) rather than apoptosis. Levels of p16 werereduced (compared to normal cells) or virtually undetectable in cellsthat constitutively overexpressed AAH; a deletion mutation of the p16gene has been correlated with invasive growth and more rapid progressionof malignant neoplasms, including those of CNS origin. These dataindicate that p16 expression is modulated by AAH.

EXAMPLE 3

[0136] Increased HAAH Production and IRS-mediated Signal Transduction

[0137] IRS-1 mediated signal transduction pathway is activated in 95% ofhuman HCC tumors compared to the adjacent uninvolved liver tissue. HAAHis a downstream effector gene involved in this signal transductionpathway. HAAH gene upregulation is closely associated withoverexpression of IRS-1 in HCC tumors as revealed by immunohistochemicalstaining and Western blot analysis. A high level of HAAH protein isexpressed in HCC and cholangiocarcinoma compared to normal hepatocytesand bile ducts. Both of these tumors also exhibit high level expressionof IRS-1 by immunohistochemical staining. FOCUS HCC cell clones stablytransfected with a C-terminal truncated dominant negative mutant ofIRS-1, which blocks insulin and IGF-1 stimulated signal transduction,was associated with a striking reduction in HAAH gene expression inliver. In contrast, transgenic mice overexpressing IRS-1 demonstrate anincrease in HAAH gene expression by Western blot analysis. Insulinstimulation of FOCUS HCC cells (20 and 40 U) in serum free medium andafter 16 hr of serum starvation demonstrated upregulation of HAAH geneexpression. These data indicate that HAAH gene expression is adownstream effector of the IRS-1 signal transduction pathway.

EXAMPLE 4

[0138] Effects of HAAH Expression Levels on the Characteristics of theMalignant Phenotype

[0139] Overexpression of IRS-1 in NIH 3T3 cells induces transformation.The full-length murine HAAH construct was cloned into the pcDNA3eukaryotic expression vector. A second murine construct encoded HAAHwith abolished catalytic activity due to a site directed mutation. Thefull-length human HAAH cDNA was cloned into the pcDNA3 expression vectoras well as a plasmid that encodes v-src which was used as a positivecontrol for transformation activity. Standard methods were used fortransfection of NIH 3T3 cells, control for transfection efficiency,assays of HAAH enzymatic activity, transformation by analysis of fociformation, anchorage-independent cell growth assays and analysis oftumorigenicity in nude mice. The data indicatet hat HAAH overexpressionis associated with generation of a malignant phenotype. TABLE 4Overexpression of enzymatically active HAAH indicates malignancy # offoci ± # of cDNA S.D.^(b) NIH 3T3 clone colonies^(e) pcDNA3  6.0 ± 3.3pcDNA 0.4 ± 0.5 (mock) (mock) murine 14.0 ± 2.9 clone 18^(d) 6.2 ± 2.9HAAH mutant  1.6 ± 1.0 clone 16^(e) 4.7 ± 6.5 murine HAAH^(a) human 32.0± 5.4 HAAH v-scr 98.0 ± 7.1

[0140] These data indicate that overexpression of HAAH is associatedwith formation of transformed foci. Enzymatic activity is required forcellular transformation to occur. Cloned NIH 3T3 cell lines withincreased human HAAH gene expression grew as solid tumors in nude mice.HAAH is a downstream effector gene of the IRS-1 signal transductionpathway.

EXAMPLE 5

[0141] Inhibition of HAAH Gene Expression

[0142] The FOCUS HCC cell line from which the human HAAH gene wasinitially cloned has a level of HAAH expression that is approximately3-4 fold higher than that found in normal liver. To make an HAAHantisense construct, the full length human HAAH cDNA was inserted in theopposite orientation into a retroviral vector containing a G418resistant gene, and antisense RNA was produced in the cells. ShorterHAAH antisense nucleic acids, e.g., those corresponding to exon 1 of theHAAH gene are also used to inhibit HAAH expression.

[0143] FOCUS cells were infected with this vector and the level of HAAHwas determined by Western blot analysis. A reduction in HAAH geneexpression was observed. Growth rate and morphologic appearance of cellsinfected with a retrovirus containing a nonrelevant Green FluorescentProtein (GFP) also inserted in the opposite orientation as a control(FIG. 8). Cells (harboring the HAAH antisense construct) exhibited asubstantial change in morphology characterized by an increase in thecytoplasm to nuclear ratio as well as assuming cell shape changes thatwere reminiscent of normal adult hepatocytes in culture. Cells withreduced HAAH levels grew at a substantially slower rate than retroviralinfected cells expressing antisense (GFP) (control) as shown in FIG. 8.A reduction in HAAH gene expression was associated with a moredifferentiated noncancerous “hepatocyte like” phenotype. Expression ofHAAH antisense sequences are used to inhibit tumor growth rate.Reduction of HAAH cellular levels results in a phenotype characterizedby reduced formation of transformed foci, low level or absent anchorageindependent growth in soft agar, morphologic features of differentiatedhepatocytes as determined by light and phase contrast microscopy, and notumor formation (as tested by inoculating the cells into nude mice).

EXAMPLE 6

[0144] Human IRS-1 Mutants

[0145] Insulin/IGF-1 stimulated expression of HAAH in HCC cell lines.Dominant-negative IRS-1 cDNAs mutated in the plextrin andphosphotryosine (PTB) domains, and Grb2, Syp and PI3K binding motifslocated in the C-terminus of the molecule were constructed. Human IRS-1mutant constructs were generated to evaluate how HAAH gene expression isupregulated by activation of the IRS-1 growth factor signal transductioncascade. Specific mutations in the C terminus of the hIRS-1 moleculeabolished the various domains which bind to SH2-effector proteins suchas Grb2, Syp and PI3K. The human IRS-1 protein contains the same Grb2and Syp binding motifs of 897YVNI (underlined in Table 5, below and1180YIDL (underlined in Table 5, below), respectively, as the rat IRS-1protein. Mutants of hIRS-1 were constructed by substitution of a TATcodon (tyrosine) with a TTT codon (phenylalanine), in these motifs byuse of oligonucleotide-directed mutagenesis suing the following primers:(5′-GGGGGAATTTGTCAATA-3′ (SEQ ID NO:8) and 5′-GAATTTGTTAATATTG-3′ (SEQID NO:9), respectively). The cDNAs of hIRS-1 (wild-type) and mutants(tyrosine 897-to-phenylalanine and tyrosine 1180-to-phenylalanine) weresubcloned into the pBK-CMV expression vector and designated ashIRS-1-wt, 897F, Δ-Grb2), 1180F, and ΔSyp. Table 5 Human IRS-1 aminoacid sequence MASPPESDGF SDVRKVGYLR KPKSMHKRFF VLRAASEAGG PARLEYYENEKKWRHKSSAP 61 (SEQ ID NO:5; KRSIPLESCF NINKRADSKN KHLVALYTRD EHFAIAADSEAEQDSWYQAL LQLHNRAKGH 121 HDGAAALGAG GGGGSCSGSS GLGEAGEDLS YGDVPPGPAFKEVWQVILKP KGLGQTKNLI 181 GIYRLCLTSK TISFVKLNSE AAAVVLQLMN IRRCGHSENFFFIEVGRSAV TGPGEFWNQV 241 DDSVVAQNMH ETILEAMRAM SDEFRPRSKS QSSSNCSNPISVPLRRHHLN NPPPSQVGLT 301 RRSRTESITA TSPASMVGGK PGSFRVRASS DGEGTMSRPASVDGSPVSPS TNRTHAHRHR 361 GSARLHPPLN HSRSIPMPAS RCSPSATSPV SLSSSSTSGHGSTSDCLFPR RSSASVSGSP 421 SDGGFISSDE YGSSPCDFRS SFRSVTPDSL GHTPPARGEEELSNYICMGG KGPSTLTAPN 481 GHYILSRGGN GHRCTPGTGL GTSPALAGDE AASAADLDNRFRKRTHSAGT SPTITHQKTP 541 SQSSVASIEE YTEMNPAYPP GGGSGGRLPG HRHSAFVPTRSYPEEGLEMH PLERRGGHHR 601 PDSSTLHTDD GYMPMSPGVA PVPSGRKGSG DYMPMSPKSVSAPQQIINPI RRHPQRVDPN 661 GYMMMSPSGG CSPDIGGGPS SSSSSSNAVP SGTSYGKLWTNGVGGHHSHV LPHPKPPVES 721 SGGKLLPCTG DYMNMSPVGD SNTSSPSDCY YGPEDPQHKPVLSYYSLPRS FKHTQRPGEP 781 EEGARHQHLR LSTSSGRLLY AATADDSSSS TSSDSLGGGYCGARLEPSLP HPHHQVLQPH 841 LPRKVDTAAQ TNSRLARPTR LSLGDPKAST LPRAREQQQQQQPLLHPPEP KSPGEYVNIE 901 FGSDQSGYLS GPVAFHSSPS VRCPSQLQPA PREEETGTEEYMKMDLGPGR RAAWQESTGV 961 EMGRLGPAPP GAASICRPTR AVPSSRGDYM TMQMSCPRQSYVDTSPAAPV SYADMRTGIA 1021 AEEVSLPRAT MAAASSSSAA SASPTGPQGA AELAAHSSLLGGPQGPGGMS AFTRVNLSPN 1081 RNQSAKVIRA DPQGCRRRHS SETFSSTPSA TRVGNTVPFGAGAAVGGGGG SSSSSEDVKR 1141 HSSASFENVW LRPGELGGAP KEPAKLCGAA GGLENGLNYIDLDLVKDFKQ CPQECTPEPQ 1201 PPPPPPPHQP LGSGESSSTR RSSEDLSAYA SISFQKQPEDRQ

[0146] Accession No. JS0670; pleckstrin domain spans residues 11-113,inclusive; Phosphate-binding residues include 46, 465, 551, 612, 632,662, 732, 941, 989, or 1012 of SEQ ID NO:5) Table 6 +HZ,52 Human IRS-1cDNA cggcggcgcg gtcggagggg gccggcgcgc agagccagac gccgccgctt gttttggttg61 (SEQ ID NO:6; gggctctcgg caactctccg aggaggagga ggaggaggga ggaggggagaagtaactgca 121 gcggcagcgc cctcccgagg aacaggcgtc ttccccgaac ccttcccaaacctcccccat 181 cccctctcgc ccttgtcccc tcccctcctc cccagccgcc tggagcgaggggcagggatg 241 agtctgtccc tccggccggt ccccagctgc agtggctgcc cggtatcgtttcgcatggaa 301 aagccacttt ctccacccgc cgagatgggc ccggatgggg ctgcagaggacgcgcccgcg 361 ggcggcggca gcagcagcag cagcagcagc agcaacagca acagccgcagcgccgcggtc 421 tctgcgactg agctggtatt tgggcggctg gtggcggctg ggacggttggggggtgggag 481 gaggcgaagg aggagggaga accccgtgca acgttgggac ttggcaacccgcctccccct 541 gcccaaggat atttaatttg cctcgggaat cgctgcttcc agaggggaactcaggaggga 601 aggcgcgcgc gcgcgcgcgc tcctggaggg gcaccgcagg gacccccgactgtcgcctcc 661 ctgtgccgga ctccagccgg ggcgacgaga gatgcatctt cgctccttcctggtggcggc 721 ggcggctgag aggagacttg gctctcggag gatcggggct gccctcaccccggacgcact 781 gcctccccgc cggcgtgaag cgcccgaaaa ctccggtcgg gctctctcctgggctcagca 841 gctgcgtcct ccttcagctg cccctccccg gcgcgggggg cggcgtggatttcagagtcg 901 gggtttctgc tgcctccagc cctgtttgca tgtgccgggc cgcggcgaggagcctccgcc 961 ccccacccgg ttgtttttcg gagcctccct ctgctcagcg ttggtggtggcggtggcagc 1021 atggcgagcc ctccggagag cgatggcttc tcggacgtgc gcaaggtgggctacctgcgc 1081 aaacccaaga gcatgcacaa acgcttcttc gtactgcgcg cggccagcgaggctgggggc 1141 ccggcgcgcc tcgagtacta cgagaacgag aagaagtggc ggcacaagtcgagcgccccc 1201 aaacgctcga tcccccttga gagctgcttc aacatcaaca agcgggctgactccaagaac 1261 aagcacctgg tggctctcta cacccgggac gagcactttg coatcgcggcggacagcgag 1321 gccgagcaag acagctggta ccaggctctc ctacagctgc acaaccgtgctaagggccac 1381 cacgacggag ctgcggccct cggggcggga ggtggtgggg gcagctgcagcggcagctcc 1441 ggccttggtg aggctgggga ggacttgagc tacggtgacg tgcccccaggacccgcattc 1501 aaagaggtct ggcaagtgat cctgaagccc aagggcctgg gtcagacaaagaacctgatt 1561 ggtatctacc gcctttgcct gaccagcaag accatcagct tcgtgaagctgaactcggag 1621 gcagcggccg tggtgctgca gctgatgaac atcaggcgct gtggccactcggaaaacttc 1681 ttcttcatcg aggtgggccg ttctgccgtg acggggcccg gggagttctggatgcaggtg 1741 gatgactctg tggtggccca gaacatgcac gagaccatcc tggaggccatgcgggccatg 1801 agtgatgagt tccgccctcg cagcaagagc cagtcctcgt ccaactgctctaaccccatc 1861 agcgtccccc tgcgccggca ccatctcaac aatcccccgc ccagccaggtggggctgacc 1921 cgccgatcac gcactgagag catcaccgcc acctccccgg ccagcatggtgggcgggaag 1981 ccaggctcct tccgtgtccg cgcctccagt gacggcgaag gcaccatgtcccgcccagcc 2041 tcggtggacg gcagccctgt gagtcccagc accaacagaa cccacgcccaccggcatcgg 2101 ggcagcgccc ggctgcaccc cccgctcaac cacagccgct ccatccccatgccggcttcc 2161 cgctgctcgc cttcggccac cagcccggtc agtctgtcgt ccagtagcaccagtggccat 2221 ggctccacct cggattgtct cttcccacgg cgatctagtg cttcggtgtctggttccccc 2281 agcgatggcg gtttcatctc ctcggatgag tatggctcca gtccctgcgatttccggagt 2341 tccttccgca gtgtcactcc ggattccctg ggccacaccc caccagcccgcggtgaggag 2401 gagctaagca actatatctg catgggtggc aaggggccct ccaccctgaccgcccccaac 2461 ggtcactaca ttttgtctcg gggtggcaat ggccaccgct gcaccccaggaacaggcttg 2521 ggcacgagtc cagccttggc tggggatgaa gcagccagtg ctgcagatctggataatcgg 2581 ttccgaaaga gaactcactc ggcaggcaca tcccctacca ttacccaccagaagaccccg 2641 tcccagtcct cagtggcttc cattgaggag tacacagaga tgatgcctgcctacccacca 2701 ggaggtggca gtggaggccg actgccggga cacaggcact ccgccttcgtgcccacccgc 2761 tcctacccag aggagggtct ggaaatgcac cccttggagc gtcggggggggcaccaccgc 2821 ccagacagct ccaccctcca cacggatgat ggctacatgc ccatgtccccaggggtggcc 2881 ccagtgccca gtggccgaaa gggcagtgga gactatatgc ccatgagccccaagagcgta 2941 tctgccccac agcagatcat caatcccatc agacgccatc cccagagagtggaccccaat 3001 ggctacatga tgatgtcccc cagcggtggc tgctctcctg acattggaggtggccccagc 3061 agcagcagca gcagcagcaa cgccgtccct tccgggacca gctatggaaagctgtggaca 3121 aacggggtag ggggccacca ctctcatgtc ttgcctcacc ccaaacccccagtggagagc 3181 agcggtggta agctcttacc ttgcacaggt gactacatga acatgtcaccagtgggggac 3241 tccaacacca gcagcccctc cgactgctac tacggccctg aggacccccagcacaagcca 3301 gtcctctcct actactcatt gccaagatcc tttaagcaca cccagcgccccggggagccg 3361 gaggagggtg cccggcatca gcacctccgc ctttccacta gctctggtcgccttctctat 3421 gctgcaacag cagatgattc ttcctcttcc accagcagcg acagcctgggtgggggatac 3481 tgcggggcta ggctggagcc cagccttcca catccccacc atcaggttctgcagccccat 3541 ctgcctcgaa aggtggacac agctgctcag accaatagcc gcctggcccggcccacgagg 3601 ctgtccctgg gggatcccaa ggccagcacc ttacctcggg cccgagagcagcagcagcag 3661 cagcagccct tgctgcaccc tccagagccc aagagcccgg gggaatatgtcaatattgaa 3721 tttgggagtg atcagtctgg ctacttgtct ggcccggtgg ctttccacagctcaccttct 3781 gtcaggtgtc catcccagct ccagccagct cccagagagg aagagactggcactgaggag 3841 tacatgaaga tggacctggg gccgggccgg agggcagcct ggcaggagagcactggggtc 3901 gagatgggca gactgggccc tgcacctccc ggggctgcta gcatttgcaggcctacccgg 3961 gcagtgccca gcagccgggg tgactacatg accatgcaga tgagttgtccccgtcagagc 4021 tacgtggaca cctcgccagc tgcccctgta agctatgctg acatgcgaacaggcattgct 4081 gcagaggagg tgagcctgcc cagggccacc atggctgctg cctcctcatcctcagcagcc 4141 tctgcttccc cgactgggcc tcaaggggca gcagagctgg ctgcccactcgtccctgctg 4201 gggggcccac aaggacctgg gggcatgagc gccttcaccc gggtgaacctcagtcctaac 4261 cgcaaccaga gtgccaaagt gatccgtgca gacccacaag ggtgccggcggaggcatagc 4321 tccgagactt tctcctcaac acccagtgcc acccgggtgg gcaacacagtgccctttgga 4381 gcgggggcag cagtaggggg cggtggcggt agcagcagca gcagcgaggatgtgaaacgc 4441 cacagctctg cttcctttga gaatgtgtgg ctgaggcctg gggagcttgggggagccccc 4501 aaggagccag ccaaactgtg tggggctgct gggggtttgg agaatggtcttaactacata 4561 gacctggatt tggtcaagga cttcaaacag tgccctcagg agtgcacccctgaaccgcag 4621 cctcccccac ccccaccccc tcatcaaccc ctgggcagcg gtgagagcagctccacccgc 4681 cgctcaagtg aggatttaag cgcctatgcc agcatcagtt tccagaagcagccagaggac 4741 cgtcagtagc tcaactggac atcacagcag aatgaagacc taaatgacctcagcaaatcc 4801 tcttctaact catgggtacc cagactctaa atatttcatg attcacaactaggacctcat 4861 atcttcctca tcagtagatg gtacgatgca tccatttcag tttgtttactttatccaatc 4921 ctcaggattt cattgactga actgcacgtt ctatattgtg ccaagcgaaaaaaaaaaatg 4981 cactgtgaca ccagaataat gagtctgcat aaacttcatc ttcaaccttaaggacttagc 5041 tggccacagt gagctgatgt gcccaccacc gtgtcatgag agaatgggtttactctcaat 5101 gcattttcaa gatacatttc atctgctgct gaaactgtgt acgacaaagcatcattgtaa 5161 attatttcat acaaaactgt tcacgttggg tggagagagt attaaatatttaacataggt 5221 tttgatttat atgtgtaatt ttttaaatga aaatgtaact tttcttacagcacatctttt 5281 ttttggatgt gggatggagg tatacaatgt tctgttgtaa agagtggagcaaatgcttaa 5341 aacaaggctt aaaagagtag aatagggtat gatccttgtt ttaagattgtaattcagaaa 5401 acataatata agaatcatag tgccatagat ggttctcaat tgtatagttatatttgctga 5461 tactatctct tgtcatataa acctgatgtt gagctgagtt ccttataagaattaatctta 5521 attttgtatt ttttcctgta agacaatagg ccatgttaat taaactgaagaaggatatat 5581 ttggctgggt gttttcaaat gtcagcttaa aattggtaat tgaatggaagcaaaattata 5641 agaagaggaa attaaagtct tccattgcat gtattgtaaa cagaaggagatgggtgattc 5701 cttcaattca aaagctctct ttggaatgaa caatgtgggc gtttgtaaattctggaaatg 5761 tctttctatt cataataaac tagatactgt tgatctttta aaaaaaaaaaaaaaaaaaaa 5821 aaaaaaaa

[0147] The double mutation of tyrosine 897 and 1180 was constructed byreplacement of 3′-sequences coding 897F by the same region of 1180Fusing restriction enzymes NheI and EcoRI, and this construct was called897F1180F orΔGrb2 ΔSyp. The expression plasmids were under control of aCMV promoter (hIRS-1-wt, ΔGrb2, ΔSyp, ΔGrb2, ΔSyp and pBK-CMV (mock) andlinearized at the 3′-end of poly A signal sequences by MluI restrictionenzymes followed by purification. A similar approach was used to changethe tyrosine residue to phenyalanine at positions 613 and 942 to createthe double PI3K mutant construct (ΔPI3K). The hIRS-1 mutants have a FLAGepitope (DYKDDDDK (SEQ ID NO:6)+stop codon) added to the C-terminus byPCR. This strategy allows to distinguish the mutant protein from “wildtype” hIRS-1 in stable transfected cell lines. The mutants are used todefine the link between the IRS signal transduction pathway andactivation of HAAH as a downstream effector gene and identify compoundsto inhibit transduction along the pathway to inhibit growth of tumorscharacterized by HAAH overexpression. Antibodies or other compoundswhich bind to phosphorylation sites or inhibit phosphorylation at thosesites are used to inhibit signal transduction and thus proleferation ofHAA-overexpressing tumors.

[0148] Other embodiments are within the following claims.

What is claimed is:
 1. A method for diagnosing a malignant neoplasm in amammal, comprising contacting a bodily fluid from said mammal with anantibody which binds to an human aspartyl (asparaginyl) beta-hydroxylase(HAAH) polypeptide under conditions sufficient to form anantigen-antibody complex and detecting the antigen-antibody complex. 2.The method of claim 1, wherein said neoplasm is derived from endodermaltissue.
 3. The method of claim 1, wherein said neoplasm is selected fromthe group consisting of colon cancer, breast cancer, pancreatic cancer,liver cancer, and cancer of the bile ducts.
 4. The method of claim 1,wherein said neoplasm is a cancer of the central nervous system (CNS).5. The method of claim 1, wherein said bodily fluid is selected from thegroup consisting of a CNS-derived bodily fluid, blood, serum, urine,saliva, sputum, lung effusion, and ascites fluid.
 6. The method of claim1, wherein said antibody is a monoclonal antibody.
 7. The method ofclaim 6, wherein said monoclonal antibody is FB50.
 8. The method ofclaim 6, wherein said monoclonal antibody is selected from the groupconsisting of 5C7, 5E9, 19B, 48A, 74A, 78A, 86A.
 9. A method forprognosis of a malignant neoplasm of a mammal, comprising (a) contactinga bodily fluid from said mammal with an antibody which binds to an HAAHpolypeptide under conditions sufficient to form an antigen-antibodycomplex and detecting the antigen-antibody complex; (b) quantitating theamount of complex to determine the level of HAAH in said fluid; and (c)comparing the level of HAAH in said fluid with a normal control level ofHAAH, wherein increasing levels of HAAH over time indicates an adverseprognosis.
 10. A method of inhibiting tumor growth in a mammalcomprising administering to said mammal a compound which inhibitsexpression of HAAH.
 11. The method of claim 10, wherein said compound isa HAAH antisense nucleic acid.
 12. The method of claim 10, wherein saidcompound is a ribozyme.
 13. The method of claim 10, wherein said tumoris derived from endodermal tissue.
 14. The method of claim 10, whereinsaid tumor is selected from the group consisting of colon cancer, breastcancer, pancreatic cancer, liver cancer, and cancer of the bile ducts.15. The method of claim 10, wherein said tumor is a CNS tumor.
 16. Amethod of inhibiting tumor growth in a mammal comprising administeringto said mammal a compound which inhibits an enzymatic activity of HAAH.17. The method of claim 16, wherein said enzymatic activity ishydroxylase activity.
 18. The method of claim 16, wherein said compoundis a dominant negative mutant of HAAH.
 19. The method of claim 18,wherein said dominant negative mutant HAAH comprises a mutation in acatalytic domain of HAAH.
 20. The method of claim 16, wherein saidcompound is an HAAH-specific intrabody.
 21. The method of claim 16,wherein said compound is L-mimosine.
 22. The method of claim 16, whereinsaid compound is a hydroxypyridone.
 23. A method of inhibiting tumorgrowth in a mammal comprising administering to said mammal a compoundwhich inhibits signal transduction through the IRS signal transductionpathway.
 24. The method of claim 23, wherein said compound inhibits IRSphosphorylation.
 25. The method of claim 23, wherein said compoundinhibits binding of Fos or Jun to an HAAH promoter sequence.
 26. Amethod of inhibiting tumor growth in a mammal comprising administeringto said mammal a compound which inhibits HAAH hydroxylation of a NOTCHpolypeptide.
 27. The method of claim 26, wherein said compound inhibitshydroxylation of an EGF-like repeat sequence in a NOTCH polypeptide. 28.A method of killing a tumor cell comprising contacting said tumor cellwith cytotoxic agent linked to an HAAH-specific antibody.
 29. Amonoclonal antibody that binds to an epitope of HAAH.
 30. The antibodyof claim 29, wherein said epitope is within a catalytic site of HAAH.31. The antibody of claim 29, wherein said monoclonal antibody isselected from the group consisting of 5C7, 5E9, 19B, 48A, 74A, 78A, 86A.32. The antibody of claim 29, wherein said monoclonal antibody isselected from the group consisting of HA238A, HA221, HA239, HA241,HA329, or HA355.
 33. A composition comprising a monoclonal antibody thatbinds to an epitope of HAAH linked to a cytotoxic agent, wherein saidcomposition preferentially kills tumor cells compared to non-tumorcells.
 34. A kit for diagnosis of a tumor in a mammal, comprising theantibody of claim
 29. 35. The kit of claim 34, wherein said antibody isimmobilized on a solid phase.
 36. The kit of claim 35, wherein saidsolid phase is selected from a group consisting of an assay plate, anassay well, a nitrocellulose membrane, a bead, a dipstick, and acomponent of an elution column.
 37. A method of determining whether acandidate compound inhibits HAAH enzymatic activity, comprising (a)providing a HAAH polypeptide; (b) providing a polypeptide comprising anEGF-like domain; (c) contacting said HAAH polypeptide or said NOTCHpolypeptide with said candidate compound; (d) determining hydroxylationof said polypeptide of step (b), wherein a decrease in hydroxylation inthe presence of said candidate compound compared to that in the absenceof said compound indicates that said compound inhibits HAAH enzymaticactivity.
 38. A method of determining whether a candidate compoundinhibits HAAH activation of NOTCH, comprising (a) providing a cellexpressing HAAH; (b) contacting said cell with a candidate compound; and(c) measuring translocation of activated NOTCH to the nucleus of saidcell, wherein a decrease in translocation in the presence of saidcompound compared to that in the absence of said compound indicates thatsaid compound HAAH activation of NOTCH.